Fibrin-binding peptides and conjugates thereof

ABSTRACT

Fibrin-binding peptides having high binding affinity and excellent physical characteristics compared to previously known fibrin-binding peptides are provided. These fibrin-binding peptides may be conjugated to a detectable label or a therapeutic agent and used to detect and facilitate treatment of pathological conditions associated with the presence of fibrin such as thrombic, angiogenic and neoplastic conditions. These peptides may be used in imaging processes such as MRI, ultrasound and nuclear medicine imaging (e.g. PET, scintigraphic imaging, etc.). The peptides may also be used therapeutically. The present invention also provides processes and methods for making and using such peptides and conjugates thereof.

RELATED APPLICATIONS

This application is the United States national stage filing ofcorresponding international application number PCT/US2007/025403 filedon Dec. 11, 2007, which claims priority to and benefit of U.S.Provisional Application No. 60/869,472, filed Dec. 11, 2006, thecontents of which are hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to fibrin-binding peptides, polypeptidesand compositions for the detection and treatment of pathologicalconditions associated with fibrin deposition or accumulation, such asintravascular thrombosis and conditions associated with angiogenicprocesses. The invention includes compounds for diagnostic and/ortherapeutic applications comprising fibrin-binding peptides. It alsoincludes methods of making and using such peptides, compounds andcompositions.

BACKGROUND OF THE INVENTION

Thrombus associated diseases are vascular conditions that develop due tothe presence of a clot. Such diseases are a major cause of mortality,and therefore developing thrombus-specific diagnosis, treatment, anddetection methodologies and reagents is of great clinical importance.Pulmonary embolism (PE), deep-vein thrombosis (DVT), stroke, andatherosclerosis are examples of thrombus-associated diseases.

DVT is a condition in which blood clots form in the deep blood vesselsof the legs and groin. These clots can block the flow of blood from thelegs back to the heart. Sometimes, a piece of a clot is detached andcarried by the bloodstream through the heart to a blood vessel, where itlodges and reduces, or blocks, the flow of blood to a vascular tissue.This is called an embolism. If such a clot lodges in pulmonary bloodvessel it can be fatal.

In the United States alone an estimated 600,000 patients suffer fromPE's each year. In approximately 378,000 of these patients, PE goesundetected, and approximately 114,000 of these patients later die due tocomplications associated with the disease. This high mortality is partlydue to the absence of clinical symptoms in many cases and to thesignificant limitations associated with currently available methods ofinvestigation and detection.

Fibrin is also associated with various cancers. The existence ofheterogeneous pattern of fibrin/fibrinogen deposition in various tumortypes is a concept supported by a substantial body of correlative andindirect evidence suggesting that fibrin/fibrinogen is important intumor stoma formation (see, for instance: Costantini V, Zacharski L R.Fibrin and cancer. Thromb Haemost. 1993; 69:406; Dvorak H F. Thrombosisand cancer. Hum Pathol. 1987; 18:275; Dvorak H F, Nagy J A, Berse B, etal. Vascular permeability factor, fibrin, and the pathogenesis of tumorstroma formation, Ann NY Acad Sci. 1992; 667:101; Cavanagh P G, Sloane BF, Honn K V. Role of the coagulation system in tumor-cell-inducedplatelet aggregation and metastasis. Hemostasis. 1988; 18:37 and BardosH, Molnar P, Csecsei G, Adany R. Fibrin deposition in primary andmetastatic human brain tumours. Blood Coagul Fibrinolysis. 1996; 7:536).Indeed, many significant hemostatic abnormalities have been described inpatients with cancer, including disseminated intravascular coagulation,hemorrhagic events, and migratory thrombophlebitis. Hemostaticcomplications are a common cause of death in patients with cancer. Manytumor cells possess strong procoagulant activities that promote thelocal activation of the coagulation system. Tumor-mediated activation ofthe coagulation cascade has been implicated in both the formation oftumor stroma and the promotion of hematogenous metastasis. Fibrinmatrix, moreover, is known to promote the migration of a substantialnumber of distinct cell types, including both transformed cells,macrophages, and fibroblasts. In particular, much like in a healingwound, the deposition of fibrin/fibrinogen, along with other adhesiveglycoproteins, into the extracellular matrix (ECM) have been shown toserve as a scaffold to support binding of growth factors and to promotethe cellular responses of adhesion, proliferation, and migration duringangiogenesis and tumor cell growth (see, for instance: Dvorak H F, NagyJ A, Berse B, et al. Vascular permeability factor, fibrin, and thepathogenesis of tumor stroma formation, Ann NY Acad Sci. 1992; 667:101;Rickles F R, Patierno S, Fernandez P M. Tissue Factor, Thrombin, andCancer. Chest. 2003; 124:58S-68S; Brown H F, Van der Water L, Hervey VS, Dvorak H F. Fibrinogen influx and accumulation of cross-linked fibrinin healing wounds and in tumor stroma. Am J Pathol. 1988; 130:4559;Dvorak H F, Hervey V S, Estrella P, Brown L F, Mc-Donagh J, Dvorak A M.Fibrin containing gels induce angiogenesis: implication for tumor stromageneration and wound healing. Lab Invest. 1987; 57:673 and Rickles F R,Patierno S, Fernandez P M. Tissue Factor, Thrombin and Cancer. Cest.2003; 124:58S-68S). Most solid tumors in humans contain considerableamounts of cross-linked fibrin, suggesting that it is important in tumorstroma formation. Studies indicate that both fibrinogen and fibrinlocalize at the tumor-host cell interface (see, for instance: Rickles FR, Patierno S, Fernandez P M. Tissue Factor, Thrombin and Cancer. Cest.2003; 124:58S-68S; Costantini V, Zacharski L R, Memoli V A et al.Fibrinogen deposition without thrombin generation in primary humanbreast cancer. Cancer Res. 1991; 51: 349-353 and Simpson-Haidaris P Jand Rybarczyky B. Tumors and Fibrinogen: The Role of Fibrinogen as anExtracellular Matrix Protein. Ann. N.Y. Acad. Sci., 2001 936(1):406-425). Fibrin matrices promote neovascularization, supporting thenotion that fibrin may facilitate tumor stroma formation by mechanismsthat are analogous to wound repair.

Moreover, a correlation seems to exist between plasma fibrinogen levelsand tumor size, depth of tumor invasion and metastasis (See, forinstance, Lee J H, Ryu K W, Kim S, Bae J M. Preoperative plasmafibrinogen levels in gastric cancer patients correlate with extent oftumor. Hepatogastroenterology 2004; 51:1860-3). In addition, it is knownthat fibrin/platelets are involved in protecting tumor cells from theaction of the circulating natural killers units provided by human immunesystem thus improving the survival of circulating tumor (See, forinstance, Palumbo J S, et al. platelets and fibrin(ogen) increasemetastatic potential by impeding natural killer cell-mediatedelimination of tumor cells. Blood, 2005; 105:178). This implies, forexample, that a conventional tumor therapy using antibodies that targettumors may not effectively treat tumors containing fibrin because thesetumor are protected by fibrin.

Thus, visualization of fibrin deposition and targetedinhibition/destruction of established vasculature and clotted fibrin isconsidered an important tool against malignant disease progression.Consequently, there remains a need for improved fibrin-binding compoundsfor use in sensitive diagnosis and specific therapy of pathologicalconditions associated with fibrin deposition, and, particularly, ofsolid tumors.

Fibrin also has been implicated in angiogenic processes. In a developingembryo, the primary vascular network is established by in situdifferentiation of meso-dermal cells in a process called vasculogenesis.After embryonic vasculogenesis it is believed that all subsequentgeneration of new blood vessels, in the embryo or in adults, is governedby the sprouting or splitting of new capillaries from the pre-existingvasculature in a process called angiogenesis (Pepper, M. et al., 1996.Enzyme Protein, 49:138-162; Risau, W., 1997. Nature, 386:671-674).Angiogenesis is not only involved in embryonic development and normaltissue growth and repair, it is also involved in the female reproductivecycle, establishment and maintenance of pregnancy, and in repair ofwounds and fractures.

In addition to normal angiogenic processes, angiogenic events also areinvolved in a number of important pathological processes, notably tumorgrowth and metastasis, and other conditions in which blood vesselproliferation is increased, such as diabetic retinopathy, psoriasis,arthropathies and rheumatoid arthritis. Indeed, angiogenesis is soimportant in the transition of a tumor from hyperplastic to neoplasticgrowth, that inhibition of angiogenesis has shown promise as a cancertherapy (Kim, K. et al., 1993. Nature, 362:841-844). In thesepathological processes, fibrin provides the structural mesh required forthe generation of new blood vessels.

There is a need, therefore, for sensitive and effective assays to detectthe presence of fibrin and fibrin-associated diseases. More specificallythere is a need for non-invasive reagents that can specifically bindfibrin and can be used to detect pathological thrombic conditions aswell as conditions associated with pathological angiogenic processes.

SUMMARY OF THE INVENTION

In answer to the need for improved materials and methods for detecting,localizing, measuring and treating fibrin clots, and pathologicalprocesses associated with fibrin, we have now surprisingly discoveredseveral non-naturally occurring polypeptides that exhibit anunexpectedly high degree of fibrin-specific binding. These polypeptidesare capable of superior fibrin specific binding compared to previouslyknown peptides and have improved physical properties such as solubility.

Another aspect of the present invention relates to modifications of theforegoing peptides to provide fibrin specific imaging agents byconjugation to a detectable label. For example, compounds in which afibrin-binding peptide is conjugated to a radiolabel, an enzymaticlabel, a label detectable by magnetic resonance imaging (MRI) such as MRparamagnetic chelates or microparticles, conjugation to or incorporationinto an ultrasound contrast agent such as gas-filled microvesicles (e.g.microbubbles, microparticles, microspheres, emulsions, or liposomes), orconjugation to an optical imaging agent, including optical dyes. Bindingmoieties according to the present invention are useful in anyapplication where binding, detecting or isolating fibrin or itsfragments is advantageous.

The present invention also relates to modifications of the foregoingpeptides to provide fibrin specific therapeutics by conjugation to atherapeutic agent. Such agents may include, for example, achemotherapeutic, a cytotoxic agent, a radiotherapeutic agent, atumoricidal agent, or a thrombolytic agent. In a preferred embodiment, apeptide is modified by conjugation to a radiotherapeutic agentcomprising a therapeutic radionuclide.

A particularly advantageous use of the binding moieties disclosed hereinis in a method of imaging thrombi, and pathological processes associatedwith fibrin in vivo. Such processes include, for example, pulmonaryembolism (PE), deep-vein thrombosis (DVT), stroke, atherosclerosis, andcancer, particularly solid tumors. The method entails the use of fibrinspecific binding moieties according to the invention for detectingthrombi or fibrin-associated pathological processes, where the bindingmoieties have been detectably labeled for use as imaging agents,including magnetic resonance imaging (MRI) contrast agents, x-rayimaging agents, radiopharmaceutical imaging agents, ultrasound imagingagents, and optical imaging agents.

In addition, the newly discovered fibrin binders can also be usedadvantageously to detect numerous other pathophysiologies in whichfibrin plays a role. In these cases, fibrin imaging can be a usefuldirect or surrogate marker for diagnosis or therapeutic monitoring. Forexample, peritoneal adhesions often occur after surgery or inflammatoryprocesses, and are comprised of a fibrin network, fibroblasts,macrophages, and new blood vessels. Patients suffering from rheumatoidarthritis, lupus, or septic arthritis often have bits offibrin-containing tissues called rice bodies in the synovial fluid oftheir joints. In thrombotic thrombocytopenic purpura, a type of anemia,fibrin deposits in arterioles cause turbulent blood flow, resulting instress and destruction of the red blood cells. The fibrin bindingmoieties of the instant invention can be used in the detection anddiagnosis of such fibrin-related disorders.

The fibrin specific agents can also be used to detect other conditionsincluding but not limited to hypoxia or ischemia of the heart, kidney,liver, lung, brain, or other organs, as well as the detection of tumors,diabetic retinopathy, early or high-risk atherosclerosis, and otherautoimmune and inflammatory disorders. Fibrin specific agents also canprovide both direct or surrogate markers of disease models in whichhypoxia and angiogenesis are expected to play a role. In hypoxicconditions, for example, fibrin(ogen) is expressed under the control ofhypoxia-inducible factor 1 (HIF-1).

The fibrin-binding peptides of the invention may also be usedtherapeutically to treat pathophysiologies in which fibrin plays a role,including, but not limited to, fibrin clots, tumors, hypoxia or ischemiaof various organs, pathological processes associated with angiogenesis,peritoneal adhesions, rheumatoid arthritis, lupus, septic arthritis, andthrombotic thrombocytopenic purpura. For example, the fibrin-bindingpeptides may be conjugated to an appropriate therapeutic radionuclideand used for radiotherapy, particularly to treat tumors. Additionally,the fibrin-binding peptides may be conjugated to an appropriatetherapeutic agent.

These and other aspects of the present invention will become apparentwith reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method for the preparation of a representativelinker-functionalized peptide according to the present invention.

FIG. 2 illustrates a method for the preparation of a representative5-carboxyfluorescein labeled peptide according to the present invention.

FIG. 3 illustrates a method for the preparation of a representativeDSPE-PEG200 peptide conjugate according to the present invention.

FIG. 4 illustrates a method for the preparation of a representativeDSPE-PG4-Glut peptide conjugate according to the present invention.

FIG. 5 illustrates a method for the preparation of a representativeDPPE-Glut-PG2-JJ peptide conjugate according to the present invention.

FIGS. 6 a, 6 b, 6 c, illustrate examples of preferred chelators foreither ¹¹¹In and lanthanides such as paramagnetic Gd³⁺ or radioactivelanthanides such as, for example, ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, and ¹⁶⁶Ho.

FIGS. 7 a, 7 b illustrate examples of preferred chelators of radioactivemetal ions such as ^(90m)Tc, ¹⁸⁶Re and ¹⁸⁸Re;

FIG. 8 relates to T1 weighted MRI images (Successive slices) acquired 4h post injection (25 μmol/kg of complex) of chelate complex 1 (bottom)and Reference Compound 2 (top) as described in Example 26.

DEFINITIONS

As used herein, unless otherwise specified, the term “polypeptide” isused to refer to a compound of two or more amino acids joined throughthe main chain (as opposed to side chain) by a peptide amide bond(—C(O)NH—). The term “peptide” is used interchangeably herein with“polypeptide” but is generally used to refer to polypeptides havingfewer than 25 amino acids.

The term “fibrin-derived polypeptide” refers to any subcomponent offibrin or fragment of fibrin that is immunologically cross-reactive withfibrin, including immunologically reactive fragments of the protein.

The term “binding” refers to the determination by standard assays,including those described herein, that a binding polypeptide recognizesand binds reversibly to a given target. Such standard assays include,but are not limited to equilibrium dialysis, gel filtration, and themonitoring of spectroscopic changes that result from binding.

The term “specificity” refers to a binding polypeptide having a higherbinding affinity for one target over another. The term “fibrinspecificity” refers to a fibrin binding moiety having a higher affinityfor fibrin than for an irrelevant target. Binding specificity can becharacterized by a dissociation equilibrium constant (K_(D)) or anassociation equilibrium constant (K_(a)) for the two tested targetmaterials, or can be any measure of relative binding strength.

The term “binding moiety” as used herein refers to any molecule capableof forming a binding complex with another molecule. “Fibrin bindingmoiety” is a binding moiety that forms a complex with a clot, soluble orinsoluble fibrin, or a soluble or insoluble fragment of fibrin having astructure or characteristic exhibited by fibrin but not fibrinogen.Included among such soluble or insoluble fragments of fibrin arefragments defined as “fibrin-derived” polypeptides. Fibrin-derivedpolypeptides, for the purposes of this invention will be used as acollective term for the DD, DD-dimer, and DD(E) polypeptides describedherein. Such fibrin-derived polypeptides are typically generated byproteolytic treatment of crosslinked fibrin but retain structuralfeatures unique to fibrin.

Specific fibrin-binding peptides are described herein (including, forexample, those included in Tables 1 and 2) and hybrid and chimericpeptides incorporating such peptides.

In addition to the detectable labels described further herein, thebinding polypeptides may be linked or conjugated to a therapeutic agentincluding a radiotherapeutic agent, a cytotoxic agent, a tumoricidalagent or enzyme, a thrombolytic agent or enzyme (e.g., tPA, plasmin,streptokinase, urokinase, hirudin), a liposome (e.g., loaded with atherapeutic agent such as a thrombolytic, an ultrasound appropriate gas,or both). In addition, binding polypeptides of the invention may bebound or linked to a solid support, well, plate, bead, tube, slide,filter, or dish. All such modified fibrin-binding moieties are alsoconsidered fibrin-binding moieties so long as they retain the ability tobind fibrin or fibrin-derived polypeptides.

A “labelling group” or “detectable label,” as used herein, is a group ormoiety capable of generating a signal that is detectable. In particulara labeling group or diagnostic label may generate a signal fordiagnostic imaging, such as magnetic resonance imaging, radioimaging,ultrasound imaging, x-ray imaging, light imaging, or carry a moiety suchas a radioactive metal or other entity that may be used in radiotherapyor other forms of therapy.

The terms “therapeutic agent” or “therapeutic” refer to a compound or anagent having a beneficial, therapeutic or cytotoxic effect in vivo.Therapeutic agents include those compositions referred to as, forexample, bioactive agents, cytotoxic agents, drugs, chemotherapy agents,radiotherapeutic agents, genetic material, etc.

The term “patient” as used herein refers to any mammal, especiallyhumans.

The term “pharmaceutically acceptable” carrier or excipient refers to anon-toxic carrier or excipient that can be administered to a patient,together with a compound of this invention, such that it does notdestroy the biological or pharmacological activity thereof.

The following common abbreviations are used throughout thisspecification: Ac₂O—acetic anhydride, CAN—acetonitrile, Ac—acetyl,API-ES—Atmospheric pressure ionization electrospray,BOP—benzotriazol—1-yloxy-tris(dimethylamino)-phosphoniumhexafluorophosphate, Bn—benzyl, Cbz—benzyloxycarboxyl, CF5-NHS—5carboxyfluorescein, succinimidyl ester (single isomer),Cha—2-Cyclohexyl-L-alanine, DCC—dicyclohexylcarbodiimide,DCM—Dichloromethane,Ddhh—12,26-diamino-1,11-dioxo-3,6,9,16,19,22-hexaoxahexacosanoyl,Dga—diglycolyl, 3-oxapentan-1,5-di-oyl,DIC—N,N′-diisopropylcarbodiimide, DIEA—N,N-Diisopropylethylamine,DMA—dimethylacetamide, DMF—Dimethylformamide, DMSO—Dimethyl sulfoxide,DPPE—1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine, commonly alsoidentified as dipalmitoylphosphatidylethanolamine,DPPG—1,2-Dipalmitoyl-sn-glycero-3-[phosphor-rac-(1-glycerol)] sodiumsalt, commonly also identified as dipalmitoylphosphatidylglycerol,DPPS—1,2-Dipalmitoyl-sn-glycero-phospho-L-serine, commonly alsoidentified as dipalmitoylphosphatidylserine,DSPA—1,2-Distearoyl-sn-glycero-phosphate sodium salt, commonly alsoidentified as distearoylphosphatdic acid,DSPE—1,2-Distearoyl-sn-glycerol-3-phosphoethanolamine, commonly alsoidentified as distearoylphosphatidylethanolamine,DSPE-PG4-NH₂—{1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)2000]},DPPE-PG4-NH₂—{1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)2000]},DSPE-PEG1000—distearoyl-glycero-phosphoethanolamine-N-methoxy(polyethyleneglycol)1000, DSPS—1,2-Distearoyl-sn-glycero-3-(phosphor-L-serine),commonly also identified as distearoylphosphatidylserine,DSG—disuccinimidylglutarate, EDAC, 1-ethyl3-(3-dimethylaminopropyl)carbodiimide HCl, EtOH—ethanol, Et₂O—diethylether, EtOAc—Ethyl acetate, Fmoc—9-Fluoroenylmethoxyloxycarbonyl,Ffe4—L-4-Fluorophenylalanine, F34fe—L-3,4-difluorophenylalanine,Glut—Glutaryl, pentan-1,5-di-oyl, HOAc—acetic acid,HOAt—1-hydroxy-7-azabenzotriazole, HPLC—High performance liquidchromatography, Hypt4—trans-4-hydroxy-L-proline, Fmoc-J orFmoc-Adoa—Fmoc-8-amino-3,6-dioxaoctanoic acid,HATU—N-{(Dimethylamino)-1H-1,2,3-triazolo(4,5-b)pyridine-1-ylmethylene]-N-methylenemethanaminiumhexafluorophosphate-N-oxide,HBTU—2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, HOBt—N-Hydroxybenzotriazole,ivDde—(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl,MALDI—Matrix Assisted Laser Desorption Ionization, Neg. ion—Negativeion, MeOH—methanol, MS—mass spectrum, NHS—N-hydroxysuccinimide,NMM—N-Methylmorpholine, NMP—N-Methylpyrrolidone, PEG—polyethylene glycol(if followed by a number, e.g. PEG4000, this identifies the approximatemean molecular weight of the polydispersed PEG polymer, i.e. about 4000daltons in the example), PFE—perfluoroethanol, Pip—Piperidine,Pd/C—palladium on carbon catalyst,Pd(PPh₃)₄—Tetrakis(triphenyl-phosphine)palladium(0), Pos. Ion—Positiveion, PyBOP—benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate, Pyr—pyridine, t_(R)—Retention time (minutes),Reagent B (TFA:H₂O:phenol:triisopropylsilane, 88:5:5:2),SATA—S-Acetylthiolacetyl,S(Galnac)—O-(2-Acetamido-2-deoxy-α-D-galactopyranosyl)-L-serine,SPPS—solid phase peptide synthesis, stearate—sodium stearate,Su—Succinimidyl, SuO—Succinimidyloxy, t-Bu—tert-Butyl,TEA—Triethylamine, Thf2ca—Tetrahydrofuran-2-carboxylic acid,TFA—Trifluoroacetic Acid, TIPS—Triisopropylsilane,9-fluorenylmethyloxycarbonyl (fmoc or Fmoc),Ttda—4,7,10-trioxatridecane-1,13-diamino,Tuda—3,6,9-Trioxaundecane-1,11-di-oyl, Aloc—Allyloxycarbonyl,Boc—tert-Butoxycarbonyl, DSG—Di-N-hydroxysuccinimidyl-glutarate,PEG3400-NHS—Polyethyleneglycol 3400 N-hydroxysuccinimidyl ester,Pmc—2,2,5,7,8-pentamethylchroman-6—sulfonyl, Trt—Trityl,DMAC—dimethylacetamide.

ABBREVIATIONS FOR AMINO ACIDS

Amino Acid 3-letter 1-letter alanine ala A arginine arg R asparagine asnN aspartic acid asp D cysteine cys C glutamic acid glu E glutamine gln Qglycine gly G histidine his H isoleucine ile I leucine leu L lysine lysK methionine met M phenylalanine phe F proline pro P serine ser Sthreonine thr T tryptophan trp W tyrosine tyr Y valine val V

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel binding moieties for fibrin. Suchbinding moieties make possible the efficient detection, imaging andlocalization of fibrin or fibrin-derived peptides in a solution orsystem that contains fibrin or fibrin-derived polypeptides. Inparticular, the binding moieties of this invention, when appropriatelylabeled, are useful for detecting, imaging and localizingfibrin-containing thrombi or other fibrin specific pathophysiologies,and can thus be used to form a variety of diagnostic and therapeuticagents for diagnosing and treating pathological conditions associatedwith, for example, angiogenesis, thrombosis and cancer, particularlysolid tumors. The preferred binding moieties of the present inventionbind fibrin and/or fibrin-derived polypeptides with high affinity, i.e.,acting at low, physiologically relevant concentrations, comparable toknown anti-fibrin antibodies and other fibrin-binding proteins andrepresent an improvement over previously known fibrin binding moieties.

Utilizing the techniques described below (including techniques describedin the Examples section), the polypeptides shown in Table 1 and in Table2 were unexpectedly found to have superior fibrin-specific binding alongwith excellent physical properties as compared to previously knownpeptides. In particular, substitution of Ala for Trp at position 6 of apreviously known peptide, Ac-WQPC*PWESWTFC*WDPGGGK-NH₂ (SEQ ID NO. 122),resulted in improved potency as well as a more hydrophilic peptide asdemonstrated by HPLC. The improved properties of this substitution wereunexpected as replacing either of the other Trp residues in the peptidelead to no or reduced fibrin binding.

Another method that was used to modify the peptideAc-WQPC*PWESWTF*CWDPGGGK-NH₂ (SEQ ID NO. 122), was attaching amino acidsat the N- or C-terminus. In general attachment of amino acids at theC-terminus did not dramatically change the potency of the peptides.However when polar amino acids (such as Arg) were added to theN-terminus, improvement in binding was observed. Considering theimportance of the Trp at position 1, the introduction of polarity withbeneficial effects was unexpected.

Another modification that led to improvements in potency was theintroduction of the unusual amino acid cyclohexylalanine (Cha) for Pheat position 11. Considering that substituted phenyl alanine derivativesled to weaker binding, it was not anticipated that changing to the morebulky, less aromatic residue would improve potency. The combination ofthese three modifications led to further improved potency.

Table 1 below provides for each of the fibrin-binding peptides of theinvention, its sequence, the sequence of the fibrin-binding moietyprepared and tested, analytical data for these peptides (including HPLCdata and mass spectral data) and, for most of the peptides, bindingaffinity measurements compared to a previously known fibrin-bindingpeptide having the sequence Ac-WQPC*PWESWTFC*WDPGGGK-NH₂, (SEQ ID NO.122) (relative IC₅₀=1). A relative IC₅₀ lower than 1 indicates betterbinding than the comparative peptide.

Note that the fibrin-binding moieties prepared and tested included thelinker GGGK at the C terminus and in some cases an Ac-group at the Nterminus. The invention encompasses fibrin-binding moieties with orwithout the GGGK linker and/or the Ac-group, as well as such moietieswith a different or additional linker, such as those described herein.Note that for Seq005 peptides were prepared with additional linkers:Ac-WQPCPAESWTFCWDPGSAGSK-NH₂, (Seq005-P2) (SEQ ID NO. 134) [HPLC Data:System D, t_(R) 3.53; Mass Spectral Data: Neg. ion: [M−H]:2377.8,[M−2H]/2:1188.4] including linker GSAGSK (SEQ ID NO. 137) andAc-WQPC*PAESWTFC*WDPGAGSGK-NH₂, (Seq005-P3) (SEQ ID NO. 135) [HPLC Data:System D, t_(R) 3.54; Mass Spectral Data: Neg. ion: [M−H]:2348.1,[M−2H]/2:1173.4], including linker GAGSGK (SEQ ID NO. 138). As shown inExample 21, the alternative linkers did not compromise the ability oftargeted microvesicles with Seq005 to bind to fibrin.

TABLE 1 Fibrin-binding peptides Mass Spectral Rel IC₅₀ (n = 2, PreparedPrepared HPLC Data Data Competition FP Seq. ID Sequence SequenceSequence ID (System, t_(R)) (Mode: Ions) assay) Seq005 WQPC*PAES Ac-Seq005-P B, 4.93 Neg. ion: [M − H]: 0.875 WTFC*WDP WQPC*PAESW 2189.6; [M− 2H]/2: (SEQ ID NO. 1) TFC*WDPGGG 1094.4 K-NH₂ (SEQ ID NO. 2) Seq014GPPGWQPC*P Ac- Seq014-P A, 4.70 Neg. ion: [M + Na − 2H]: 0.235 WESWTFC*WGPPGWQPC*P 2636.8; [M − 2H]/ DP (SEQ ID WESWTFC*WD 2: 1306.2 NO. 3)PGGGK-NH₂ (SEQ ID NO. 4) Seq015 GGRGWQPC* Ac- Seq015-P A, 4.44 Pos. ion:0.370 PWESWTFC* GGRGWQPC*P [2M + 3H]/3: WDP (SEQ ID WESWTFC*WD 1756.9;[M + 2H]/2: NO. 5) PGGGK-NH₂ 1317.3; [M + 3H]/3: (SEQ ID NO. 6) 876.1Seq016 GWQPC*PWE Ac- Seq016-P B, 4.08 Neg. ion: [M − H]: 1.04 SWTFC*WDPGWQPC*PWES 2160.2; [M − 2H]/2: (SEQ ID NO. 7) WTFC*WDPGG 1180.5; [M −3H]/3: GK-NH₂ (SEQ 786.8 ID NO. 8) Seq017 SGSGJWQPC* Ac- Seq017-P B,3.96 Neg. ion: [M − H]: 0.714 PWESWTFC* SGSGJWQPC*P 2738.7; [M − 2H]/2:WDP (SEQ ID WESWTFC*WD 1369.1 NO. 9) PGGGK-NH₂ (SEQ ID NO. 10) Seq018WQPC*PWES Ac- Seq018-P B, 4.27 Neg. ion: [M − H]: 0.494 WT-Cha-WQPC*PWESW 2311.8; [M − 2H]/2: C*WDP (SEQ T-Cha- 1154.8 ID NO. 11)C*WDPGGGK- NH₂ (SEQ ID NO. 12) Seq019 WQPC*PWES Ac- Seq019-P B, 4.26Neg. ion: [M − H]: 0.601 WT-Ffe4- WQPC*PWESW 2322.6; [M − 2H]/2: C*WDP(SEQ T-Ffe4- 1160.4 ID NO. 13) C*WDPGGGK- NH₂ (SEQ ID NO. 14) Seq020WQPC*PWES Ac- Seq020-P B, 4.33 Neg. ion: [M − H]: 0.428 WT-F34fe-WQPC*PWESW 2341.2; [M − 2H]/2: C*WDP (SEQ T-F34fe- 1169.8 ID NO. 15)C*WDPGGGK- NH₂ (SEQ ID NO. 16) Seq021 GWQPC*PWE GWQPC*PWES Seq021-P B,3.94 Neg. ion: [M − H]: 0.622 SWTFC*WDP WTFC*WDPGG 2319.6; [M − 2H]/2:(SEQ ID GK-NH₂ (SEQ 1159.3 NO. 17) ID NO. 18) Seq022 RGWQPC*PWRGWQPC*PWE Seq022-P B, 3.79 Pos. ion 0.511 ESWTFC*WD SWTFC*WDPG [M +2H]/2: 1239.3; P (SEQ ID GGK-NH₂ (SEQ [M + 3H]/3: 826.8 NO. 19) ID NO.20) Seq023 RWQPC*PWE RWQPC*PWES Seq023-P B, 3.77 Pos. ion 0.47 SWTFC*WDPWTFC*WDPGG [M + 2H]/2: 1211.6; (SEQ ID GK-NH₂ (SEQ [M + 3H]/3: 807.6 NO.21) ID NO. 22) Seq024 SGSGSGSGW Ac- Seq024-P B, 3.89 Neg. ion: [M − 2H]/0.527 QPC*PWESW SGSGSGSGWQ 2: 1439.8 TFC*WDP PC*PWESWTFC (SEQ ID*WDPGGGK- NO. 23) NH₂ (SEQ ID NO. 24) Seq025 KKGWQPC*P Ac- Seq025-P A,4.26 Neg. ion: [M − H]: 0.357 WESWTFC*W KKGWQPC*PW 2618.8; [M − 2H]/2:DP (SEQ ID ESWTFC*WDP 1308.1 NO. 25) GGGK-NH₂ (SEQ ID NO. 26) Seq026KGKGKGWQ Ac- Seq026-P A, 4.11 Neg. ion: [M − H]: 0.595 PC*PWESWTFKGKGKGWQP 2860.5; [M − 2H]/2: C*WDP (SEQ C*PWESWTFC* 1429.6 ID NO. 27)WDPGGGK- NH₂ (SEQ ID NO. 28) Seq027 S(Galnac)- Ac-S(Galnac)- Seq027-P B,3.95 Neg. ion: [M − H]: 0.5595 WQPC*PWES WQPC*PWESW 2595.5; [M − 2H]/2:WTFC*WDP TFC*WDPGGG 1297.0 (SEQ ID K-NH₂ (SEQ ID NO. 29) NO. 30) Seq028Thf2ca- Thf2ca- Seq028-P B, 4.23 Neg. ion: [M − H]: 0.616 WQPC*PWESWQPC*PWESW 2416.8; [M − 2H]/2: WTFC*WDP TFC*WDPGGG 1208.6 (SEQ ID K-NH₂(SEQ ID NO. 31) NO. 32) Seq029 RRGGWQPC* Ac- Seq029-P A, 4.32 Pos. ion0.125 PWESWTFC* RRGGWQPC*P [M + 2H]/2: 1366.4; WDP (SEQ ID WESWTFC*WD[M + 3H]/3: 911.8; NO. 33) PGGGK-NH₂ [M + 3H + Na]/4: (SEQ ID NO. 34)689.8 Seq031 S(Galnac)- Ac-S(Galnac)- Seq031-P B, 3.95 Neg. ion: [M −H]: 1.15 JWQPC*PWES JWQPC*PWES 2740.4; [M − 2H]/2; WTFC*WDP WTFC*WDPGG1369.3 (SEQ ID GK-NH₂ (SEQ NO. 35) ID NO. 36) Seq032 WQPC*- Ac-WQPC*-Seq032-P B, 4.12 Mode: Neg. −ion: 0.55 Hypt4- Hypt4- [M − H]: 2320.7;WESWTFC*W WESWTFC*WD [M − 2H]/2: 1159.4 DP (SEQ ID PGGGK-NH₂ NO. 37)(SEQ ID NO. 38) Seq034 GPPGWQPC*P Ac- Seq034-P D, 3.49 Neg. Ion —[M −H]: § AESWTFC*W GPPGWQPC*P 2498.9, [M − 2H]/2: DP (SEQ ID AESWTFC*WD1248.4 NO. 39) PGGGK-NH₂ (SEQ ID NO. 40) Seq035 GGRGWQPC* Ac- Seq035-PD, 3.29 Neg. Ion - [M − H]: § PAESWTFC* GGRGWQPC*P 2516.7, [M + TFA −2H]/ WDP (SEQ ID AESWTFC*WD 2: 1314.7, [M − 2H]/ NO. 41) PGGGK-NH₂ 2:1257.9 (SEQ ID NO. 42) Seq036 KKGWQPC*P Ac- Seq036-P D, 4.91 Neg. Ion -[M − H]: § AESWTFC* KKGWQPC*PA 2916.4, [2M − 3H]/ WDP (SEQ ID ESWTFC*WDP3: 1943.6, [M − 2H]/ NO. 43) GGGK-NH₂ ^(†) 2: 1457.6 (SEQ ID NO. 44)Seq037 KGKGKGWQ Ac- Seq037-P D, 5.26 Neg. Ion - [2M − 3H]/ § PC*PAESWTFKGKGKGWQP 3: 2242.8, C*WDP (SEQ C*PAESWTFC* 2225.4, [M − 2H]/2: ID NO.45) WDPGGGK- 1681.2 NH₂ ^(†) (SEQ ID NO. 46) Seq038 GWQPC*PAE GWQPC*PAESSeq038-P D, 3.76 Neg. Ion - [M − H]: § SWTFC*WDP WTFC*WDPGG 2288.6, [M −2H]/2: (SEQ ID GK-NH₂ ^(‡) (SEQ 1143.9 NO. 47) ID NO. 48) Seq039GWQPC*PAE Ac- Seq039-P D, 3.44 Neg. Ion - [M − H]: § SWTFC*WDPGWQPC*PAES 2247.9, [M − 2H]/2: (SEQ ID WTFC*WDPGG 1122.9 NO. 49) GK-NH₂(SEQ ID NO. 50) Seq040 SGSGSGSGW Ac- Seq040-P D, 3.25 Neg. Ion - [2M −3H]/ § QPC*PAESWT SGSGSGSGWQ 3: 1844.3, [M − 2H]/ FC*WDP (SEQPC*PAESWTFC 2: 1382.8 ID NO. 51) *WDPGGGK- NH₂ (SEQ ID NO. 52) Seq041WQPC*PAES Ac- Seq041-P D, 3.65 Neg. Ion - [M − H]: § WT-Ffe4- WQPC*PAESW2207.7, [M − 2H]/2: C*WDP (SEQ T-Ffe4- 1103.4 ID NO. 53) C*WDPGGGK- NH₂(SEQ ID NO. 54) Seq042 WQPC*PAES Ac- Seq042-P D, 3.69 Neg. Ion -[M − H]:§ WT-Cha- WQPC*PAESW 2195.7, [M − 2H]/2: C*WDP (SEQ T-Cha- 1097.4 ID NO.55) C*WDPGGGK- NH₂ (SEQ ID NO. 56) Seq043 WQPC*PAES Ac- Seq043-P D, 3.73Neg. Ion - [M − H]: § WT-F34fe- WQPC*PAESW 2225.4, [M − 2H]/2: C*WDP(SEQ T-F34fe- 1111.9 ID NO. 57) C*WDPGGGK- NH₂ (SEQ ID NO. 58) Seq044Thf2ca- Thf2ca- Seq044-P D, 3.71 Neg. Ion - [M − H]: § WQPC*PAESWQPC*PAESW 2245.6, [M − 2H]/2: WTFC*WDP TFC*WDPGGG 1122.3. (SEQ ID K-NH₂(SEQ ID NO. 59) NO. 60) Seq045 SGSGJWQPC* Ac- Seq045-P D, 3.34 Neg.ion - [M − 2H]/ § PAESWTFC* SGSGJWQPC*P 2: 1311.3 WDP (SEQ ID AESWTFC*WDNO. 61) PGGGK-NH₂ (SEQ ID NO. 62) Seq046 RRGGWQPC* Ac- Seq046-P D, 3.12Pos. ion - § PAESWTFC* RRGGWQPC*P [2M + 3H]/3: WDP (SEQ ID AESWTFC*WD1745.4, NO. 63) PGGGK-NH₂ [M + 2H]/2: 1309.5, (SEQ ID NO. 64) [M +3H]/3: 873.3 Seq047 RRGGWQPC*- Ac- Seq047-P G, 4.5 Mode: Pos. ion; 0.75Hypt4- RRGGWQPC*- [M + 2H]/2: 1375.0, WESWTFC*W Hypt4- [M + 3H]/3:917.3, DP (SEQ ID WESWTFC*WD [M + Na + 3H]/4: NO. 65) PGGGK-NH₂ 693.8,(SEQ ID NO. 66) [M + 2Na + 3H]/5: 558.9 Seq048 RWQPC*PWE Ac- Seq048-P B,3.72 Neg. Ion: [M − H]: § SWTFC*WDP RWQPC*PWES 2461.8, [M − 2H]/2: (SEQID WTFC*WDPGG 1230.0, [M + TFA − 2H]/ NO. 67) GK-NH₂ (SEQ 2: 1286.8 IDNO. 68) Seq049 RWQPC*PAES Ac- Seq049-P D, 3.43 Neg. ion: [M − H]: §WT-Cha- RWQPC*PAES 2352.9, [M − 2H]/2: C*WDP (SEQ WT-Cha- 1175.4, [M +TFA − 2H]/ ID NO. 69) C*WDPGGGK- 2: 1232.2 NH₂ (SEQ ID NO. 70) Seq050GWQPC*PAE GWQPC*PAES Seq050-P D, 3.92 Neg. ion: [M − H]: § SWT-Cha-WT-Cha- 2294.7, [M − 2H]/2: C*WDP (SEQ C*WDPGGGK- 1146.9 (as Aloc ID NO.71) NH₂† (SEQ ID peptide) NO. 72) Seq051 RGWQPC*PW Ac- Seq051-P D, 3.64Neg. ion: [M − H]: § ESWTFC*WD RGWQPC*PWE 2517.9, [M − 2H]/2: P (SEQ IDSWTFC*WDPG 1258.8, [M + TFA − NO. 73) GGK-NH₂ (SEQ 2H]/2: 1315.5 ID NO.74) Seq052 RGWQPC*PA Ac- Seq052-P D, 3.46 Neg. Ion: [M − H]: § ESWTFC*WDRGWQPC*PAE 2403.3, [M − 2H]/2: P (SEQ ID SWTFC*WDPG 1200.9 NO. 75)GGK-NH₂ (SEQ ID NO. 76) Seq053 RGWQPC*PA Ac- Seq053-P D, 3.48 Neg. ion:[M − H]: § ESWT-Cha- RGWQPC*PAE 2409.0, [M − 2H]/2: C*WDP (SEQ SWT-Cha-1204.1, [M + TFA- ID NO. 77) C*WDPGGGK- 2H]/2: 1261.1 NH₂ (SEQ ID NO.78) Seq054 RGWQPC*PA RGWQPC*PAE Seq054-P D, 3.91 Neg. ion: [M − H]: §ESWT-Cha- SWT-Cha- 2451.0, [M − 2H]/2: C*WDP (SEQ C*WDPGGGK- 1224.7,[M + TFA- ID NO. 79) NH₂ ^(‡) (SEQ ID 2H]/2: 1282.2 (as NO. 80) Alocpeptide) Seq055 GWQPC*PAE Ac- Seq055-P D, 3.59 Neg. ion: [[M − H]: §SWT-Cha- GWQPC*PAES 2253.1, [M − 2H]/2: C*WDP (SEQ WT-Cha- 1125.9 ID NO.81) C*WDPGGGK- NH₂ (SEQ ID NO. 82) Seq056 RWQPC*PAES RWQPC*PAES Seq056-PD, 3.71 Neg. ion: [M − H]: § WTFC*WDP WTFC*WDPGG 2388.0, [M − 2H]/2:(SEQ ID GK-NH₂‡ (SEQ 1193.4, [M + TFA- NO. 83) ID NO. 84) 2H]/2: 1250.8(as Aloc peptide) Seq057 RWQPC*PAES RWQPC*PAES Seq057-P D, 3.71 Neg.ion: [M − H]: § WT-Cha- WT-Cha- 2394.0, [M − 2H]/2: C*WDP (SEQC*WDPGGGK- 1196.4, [M + TFA- ID NO. 85) NH₂‡ (SEQ ID 2H]/2: 1252.9 (asNO. 86) Aloc peptide) †= Analytical data reported for peptide bearingthe ivDde group on N^(e) of all lysine groups of the peptide except forthe C-terminal lysine. ‡= Analytical data reported for N-terminalAloc-protected peptide. § = Direct binding assay conducted using CF5labeled peptide, see table 2

The details of the HPLC systems used are set forth in the Examplessection. Details of fibrin-binding assays are also set forth in theExamples section.

Changes from the known peptide, Ac-WQPC*PWESWTFC*WDPGGGK-NH₂ (SEQ ID NO.122), also referred to herein as Seq000, are underlined, but omissionsfrom this sequence (e.g. initial Ac, etc.) are not highlighted. Thus,for example, within the peptide of the invention coded as Seq005, theamino acid therein defined as “A” replaces, in that same position, thecorresponding amino acid “W” in the prior art peptide, Seq000.

As used in Table 1 and elsewhere herein, the designation “C*” refers toa cysteine residue that contributes to a disulfide bond.

As shown in Table 1, all of the peptides described therein haveequivalent or far superior binding than the comparative peptide.

Table 2 below provides for each of the 5-carboxyfluorescein labeledfibrin-binding peptides included therein, its sequence, HPLC data, massspectral data and, for most of the peptides, binding affinitymeasurements compared to the previously known fibrin-binding peptidehaving the sequence Ac-WQPC*PWESWTFC*WDPGGGK-NH₂(Seq000) (SEQ ID NO.122).

TABLE 2 Fibrin Binding Peptides - 5-Carboxyfluorescein Labeled PeptidesHPLC Data K_(D) (μM) (System, Mass Spectral Data Direct Binding Seq. IDSequence t_(R)) (Mode; Ions) (n = 2) Seq000- Ac- F, 14.78 Neg. ion; [M −2H]/2: 0.39 CF5 WQPC*PWESWT 1330.9; [M − 3H]/3: FC*WDPGGGK 886.9; [M −4H]/4: (CF5)-NH₂ 665.0 (SEQ ID NO. 87) Seq014- Ac- D,  4.31Neg. ion; [M − 2H]/2: 0.25 CF5 GPPGWQPC*PW 1485.2, [2M − 3]/3:ESWTFC*WDPG 1981.8 GGK(CF5)-NH₂ (SEQ ID NO. 88) Seq015- Ac- D,  4.10Neg. ion: [M − 2H]/2: 0.29 CF5 GGRGWQPC*P 1494.9, [2M − 3H]/3:WESWTFC*WDP 1993.5, [3M + Na − 5H]/ GGGK(CF5)-NH₂ 4: 2249.1(SEQ ID NO. 89) Seq016- Ac- D,  4.35 Neg. ion; [M − 2H]/2: 0.23 CF5GWQPC*PWES 1359.4, [2M − 3H]/3: WTFC*WDPGG 1812.9 GK(CF5)-NH₂(SEQ ID NO. 90) Seq017- Ac- D,  4.15 Neg. ion; [M − 2H]/2: 0.45 CF5SGSGJWQPC*P 1547.8 WESWTFC*WDP GGGK(CF5)-NH₂ (SEQ ID NO. 91) Seq018- Ac-D,  4.49 Neg. ion; [M − H]: 0.20 CF5 WQPC*PWESWT- 2670.3, [M − 2H]/2:Cha- 1333.9, [M − 3H]/3: C*WDPGGGK(CF5)- 888.7 NH₂ (SEQ ID NO. 92)Seq019- Ac- D,  4.50 Neg. ion; [M − 2H]/2: 1.02 CF5 WQPC*PWESWT- 1339.8Ffe4- C*WDPGGGK(CF5)- NH₂ (SEQ ID NO. 93) Seq020- Ac- D,  4.56Neg. ion; [2M − 3]/3: 0.70 CF5 WQPC*PWESWT- 1798.5; [M − 2H]/2: F34fe-1348.7 C*WDPGGGK(CF5)- NH₂ (SEQ ID NO. 94) Seq021- GWQPC*PWES D,  4.31Neg. ion; [M − 2H]/2: N/D CF5 WTFC*WDPGG 1338.9, [M − 3H]/3: GK(CF5)-NH₂892.0 (SEQ ID NO. 95) Seq022- RGWQPC*PWES D,  3.99 Neg. ion; [2M −3H]/3: 0.28 CF5 WTFC*WDPGG 1889.3, [M − 2H]/2: GK(CF5)-NH₂ 1416.4, [M −3H]/3: (SEQ ID NO. 96) 944.0 Seq023- RWQPC*PWES D,  4.00 Neg. ion; [M −2H]/2: 0.11 CF5 WTFC*WDPGG 1387.9, [M − 3H]/3: GK(CF5)-NH₂ 925.0(SEQ ID NO. 97) Seq024- Ac- D,  4.11 Neg. ion; [M − 2H]/2: 0.47 CF5SGSGSGSGWQP 1619.1, [2M − 3H]/3: C*PWESWTFC* 2158.5 WDPGGGK(CF5)- NH₂(SEQ ID NO. 98) Seq025- Ac- D,  3.94 Neg. ion; [M − 2H]/2: 0.71 CF5KKGWQPC*PW 1487.7, [2M − 3H]/3: ESWTFC*WDPG 1984.5 GGK(CF5)-NH₂(SEQ ID NO. 99) Seq026- Ac- D,  3.79 Neg. ion; [M − 2H]/2: 0.76 CF5KGKGKGWQPC 1608.6, [2M − 3H]/3: *PWESWTFC*W 2145.1 DPGGGK(CF5)- NH₂(SEQ ID NO. 100) Seq027- Ac-S(Galnac)- D,  4.16 Neg. ion; [M − 2H]/2:0.50 CF5 WQPC*PWESWT 1476.4, [2M − 3H]/3: FC*WDPGGGK 1968.6, [2M −6H]/6: (CF5)-NH₂ 984.5 (SEQ ID NO. 101) Seq028- Thf2ca- D,  4.13Neg. ion; [M + Na − 2H]: 1.21 CF5 WQPC*PWESWT 2740.2; [M + Na − 3H]/2:FC*WDPGGGK 1370.1 [M − 2H]/2: (CF5)-NH₂ 1359.0, [M − 3H]/3:(SEQ ID NO. 102) 905.4 Seq029- Ac- D,  4.15 Neg. ion; [M − 2H]/2: N/DCF5 RRGGWQPC*P 1543.7, [2M − 3H]/3: WESWTFC*WDP 2059.8, [M − 3H]/3:GGGK(CF5)-NH₂ 1029.0 (SEQ ID NO. 103) Seq034- Ac- D,  3.45Neg. ion; [M − H]: 0.31 CF5 GPPGWQPC*PA 2857.4; [M − 2H]/2: ESWTFC*WDPG1228.0 GGK(CF5)-NH₂ (SEQ ID NO. 104) Seq035- Ac- D,  3.88Neg. Ion; [2M − 3H]/3: 0.52 CF5 GGRGWQPC*PA 1916.3, [M − 2H]/2:ESWTFC*WDPG 1437.4 GGK(CF5)-NH₂ (SEQ ID NO. 105) Seq038- GWQPC*PAESWD,  3.87 Neg. ion; [M − 2H]/2: 0.24 CF5 TFC*WDPGGGK 1280.8 (CF5)-NH₂(SEQ ID NO. 106) Seq039- Ac- D,  4.04 Neg. ion; [M − 2H]/2: 0.24 CF5GWQPC*PAESW 1302.7 TFC*WDPGGGK (CF5)-NH₂ (SEQ ID NO. 107) Seq040- Ac-D,  3.82 Neg. ion; [2M − 3H]/3: 0.53 CF5 SGSGSGSGWQP 2083.1, [M − 2H]/2:C*PAESWTFC* 1561.8, [M − 3H]/: WDPGGGK(CF5)- 1040.7 NH₂ (SEQ ID NO. 108)Seq042- Ac- D,  4.29 Neg. ion; [M − H]: 0.17 CF5 WQPC*PAESWT-2554.6, [M − 2H]/2: Cha- 1276.9 C*WDPGGGK(CF5)- NH₂ (SEQ ID NO. 109)Seq045- Ac- D,  3.90 Neg. ion; [M − H]: 0.57 CF5 SGSGJWQPC*PA2982.0, [M − 2H]/2: ESWTFC*WDPG 1489.9 GGK(CF5)-NH₂ (SEQ ID NO. 110)Seq046- Ac- D,  3.50 Pos. ion; [M + 2H]/2: 0.22 CF5 RRGGWQPC*PA1488.9, [M + 3H]/3: ESWTFC*WDPG 992.7 GGK(CF5)-NH₂ (SEQ ID NO. 111)Seq048- Ac- D,  4.26 Neg. ion; [M − 2H]/2: 0.11 CF5 RWQPC*PWES1409.1, [2M − 3H]/3: WTFC*WDPGG 1879.1 GK(CF5)-NH₂ (SEQ ID NO. 112)Seq049- Ac- D,  3.94 Neg. ion; [M − H]: 0.21 CF5 RWQPC*PAESW2704.5, [2M − 3H]/3: TFC*WDPGGGK 1802.6, [M − 2H]/2: (CF5)-NH₂ 1351.3(SEQ ID NO.113) Seq050- Ac- D,  4.04 Neg. ion; [M − H]: 0.08 CF5RWQPC*PAESW 2710.9, [2M − 3H]/3: T-Cha- 1806.4, [M − 2H]/2: C*WDPGGGK1354.5 (CF5)-NH₂ (SEQ ID NO. 114) Seq051- GWQPC*PAESW D,  4.03Neg. ion; [M − H]: 0.06 CF5 T-Cha- 2569.5, [M − 2H]/2: C*WDPGGGK1283.8, [2M −3H]/3: (CF5)-NH₂ 1712.1 (SEQ ID NO. 115) Seq052- Ac-D,  4.17 Neg. ion; [3M − 4H]/4: 0.53 CF5 RGWQPC*PWES 2157.7, [2M −3H]/3: WTFC*WDPGG 1917.3, [M − 2H]/2: GK(CF5)-NH₂ 1437.9(SEQ ID NO. 116) Seq053- Ac- D,  4.04 Neg. ion; [M − H]: 0.10 CF5RGWQPC*PAES 2767.5, [2M − 3H]/3: WT-Cha- 1844.1, [M − 2H]/2: C*WDPGGGK1383.0 (CF5)-NH₂ (SEQ ID NO. 117) Seq054- RGWQPC*PAES D,  3.91Neg. ion; [M − H]: 0.05 CF5 WT-Cha- 2726.1, [2M − 3H]/3: C*WDPGGGK1816.9, [M − 2H]/2: (CF5)-NH₂ 1362.0 (SEQ ID NO. 118) Seq055- Ac-D,  4.20 Neg. ion; [M − H]: 0.08 CF5 GWQPC*PAESW 2611.6, [M − 2H]/2:T-Cha- 1304.8, [2M − 3H]/3: C*WDPGGGK 1740.2 (CF5)-NH₂ (SEQ ID NO. 119)Seq056- RWQPC*PAESW D,  3.81 Neg. ion; [M − H]: 0.06 CF5 TFC*WDPGGGK2663.1, [2M − 3H]/3: (CF5)-NH₂ 1774.2, [M − 2H]/2: (SEQ ID NO. 120)1330.3 Seq057- RWQPC*PAESW D,  3.91 Neg. ion; [M − H]: 0.03 CF5 T-Cha-2668.2, [M − 2H]/2: C*WDPGGGK 1333.3 (CF5)-NH₂ (SEQ ID NO. 121)

The details of the HPLC systems used are set forth in the Examplessection. Details of fibrin-binding assays are also set forth in theExamples section.

As shown in Table 2, all of the 5-carboxyfluorescein labeledfibrin-binding peptides described therein have equivalent or farsuperior binding than the comparative fibrin-binding peptide.

Table 3 below, provides residue abbreviations and the correspondingstructure of some of the commonly used residues referenced in Table 1and Table 2 above.

TABLE 3 Abbreviations for Residues Residue Residue AbbreviationStructure Abbreviation Structure Cha

S(Galnac)

Ffe4

Thf2ca

F34fe

Hypt4

Btn

CF5

Direct synthesis of the fibrin-binding peptides of the invention may beaccomplished using conventional techniques, including solid-phasepeptide synthesis, solution-phase synthesis, etc. Solid-phase synthesisis preferred. See Stewart et al., Solid-Phase Peptide Synthesis (W. H.Freeman Co., San Francisco, 1989); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963); Bodanszky and Bodanszky, The Practice of PeptideSynthesis (Springer-Verlag, New York, 1984), incorporated herein byreference.

A fibrin-binding peptide according to the present invention ispreferably purified once it has been isolated or synthesized. Forpurification purposes, there are many standard methods that may beemployed, including reversed-phase high-pressure liquid chromatography(RP-HPLC) using an alkylated silica column such as C₄-, C₈- orC₁₈-silica. A gradient mobile phase of increasing organic content isgenerally used to achieve purification, for example, acetonitrile in anaqueous buffer, usually containing a small amount of trifluoroaceticacid. Ion-exchange chromatography can also be used to separate peptidesbased on their charge. The degree of purity of the polypeptide may bedetermined by various methods, including identification of a major largepeak on HPLC. A polypeptide that produces a single peak that is at least95% of the input material on an HPLC column is preferred. Even morepreferable is a polypeptide that produces a single peak that is at least97%, at least 98%, at least 99% or even 99.5% or more of the inputmaterial on an HPLC column.

In order to ensure that the fibrin-binding peptide obtained is thedesired peptide for use in compositions of the present invention,analysis of the peptide composition may be carried out. Such compositionanalysis may be conducted using high resolution mass spectrometry todetermine the molecular weight of the peptide. Alternatively, the aminoacid content of the peptide can be confirmed by hydrolyzing the peptidein aqueous acid, and separating, identifying and quantifying thecomponents of the mixture using HPLC, or an amino acid analyzer. Proteinsequencers, which sequentially degrade the peptide and identify theamino acids in order, may also be used to determine definitely thesequence of the peptide.

The fibrin binding polypeptides of the invention may be conformationallyrestrained by disulfide linkages between the two cysteine residues intheir sequence. This conformational restraint ensures that the peptideshave a binding structure that contributes to the peptides' affinity forfibrin and their specificity for fibrin over fibrinogen. Other methodsfor constraining peptides which would retain a similar conformation andfibrin specificity for the peptide have been described in the art andmay be used herein.

Modification or Optimization of Binding Polypeptides

Modification or optimization of the fibrin-binding polypeptides iswithin the scope of the present invention. Specifically, a polypeptidesequence of the invention can be further modified to optimize itspotency, pharmacokinetic behavior, stability and/or other biological,physical and chemical properties.

Substitution of Amino Acid Residues

Substitutions of amino acids within the same class (e.g., substitutingone basic amino acid for another) are well known in the art. Forexample, one can make the following isosteric and/or conservative aminoacid changes in the parent polypeptide sequence with the expectationthat the resulting polypeptides would have a similar or improved profileof the properties described above:

Substitution of alkyl-substituted hydrophobic amino acids: Includingalanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid,S-cyclohexylalanine or other simple alpha-amino acids substituted by analiphatic side chain from 1-10 carbons including branched, cyclic andstraight chain alkyl, alkenyl or alkynyl substitutions.

Substitution of aromatic-substituted hydrophobic amino acids: Includingphenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine,2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine,histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro,chloro, bromo, or iodo) or alkoxy (from C₁-C₄)-substituted forms of theprevious listed aromatic amino acids, illustrative examples of whichare: 2-, 3-, or 4-aminophenylalanine, 2-, 3-, or 4-chlorophenylalanine,2-, 3-, or 4-methylphenylalanine, 2-, 3-, or 4-methoxyphenylalanine,5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or4′-amino-, 2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-,3′-, or 4′-methyl-2-, 3- or 4-biphenylalanine, and 2- or3-pyridylalanine.

Substitution of amino acids containing basic functions: Includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀ branched,linear, or cyclic) derivatives of the previous amino acids, whether thesubstituent is on the heteroatoms (such as the alpha nitrogen, or thedistal nitrogen or nitrogens, or on the alpha carbon, in the pro-Rposition for example. Compounds that serve as illustrative examplesinclude: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine,3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma'-diethyl-homoarginine.Included also are compounds such as alpha methyl arginine, alpha methyl2,3-diaminopropionic acid, alpha methyl histidine, alpha methylornithine where alkyl group occupies the pro-R position of the alphacarbon. Also included are the amides formed from alkyl, aromatic,heteroaromatic (where the heteroaromatic group has one or morenitrogens, oxygens or sulfur atoms singly or in combination) carboxylicacids or any of the many well-known known activated derivatives such asacid chlorides, active esters, active azolides and related derivatives)and lysine, ornithine, or 2,3-diaminopropionic acid.

Substitution of acidic amino acids: Including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, aralkyl, and heteroarylsulfonamides of 2,3-diaminopropionic acid, ornithine or lysine andtetrazole-substituted alkyl amino acids.

Substitution of side chain amide residues: Including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine.

Substitution of hydroxyl containing amino acids: Including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine.

It is also understood that the amino acids within each of the categorieslisted above may be substituted for another of the same group.

Substitution of Amide Bonds

Another type of modification within the scope of the invention is thesubstitution of amide bonds within the backbone of a bindingpolypeptide. For example, to reduce or eliminate undesired proteolysis,or other degradation pathways which diminish serum stability, resultingin reduced or abolished bioactivity, or to restrict or increaseconformational flexibility, it is common to substitute amide bondswithin the backbone of the peptides with functionality that mimics theexisting conformation or alters the conformation in the manner desired.Such modifications may produce increased binding affinity or improvedpharmacokinetic behavior. It is understood that those knowledgeable inthe art of peptide synthesis can make the following amide bond-changesfor any amide bond connecting two amino acids with the expectation thatthe resulting peptides could have the same or improved activity:insertion of alpha-N-methylamides or peptide amide backbone thioamides,removal of the carbonyl to produce the cognate secondary amines,replacement of one amino acid with an aza-aminoacid to producesemicarbazone derivatives, and use of E-olefins and substitutedE-olefins as amide bond surrogates.

Introduction of D-Amino Acids

Another approach within the scope of the invention is the introductionof D-alanine, or another D-amino acid, distal or proximal to a labilepeptide bond. In this case it is also understood to those skilled in theart that such D-amino acid substitutions can, and at times, must bemade, with D-amino acids whose side chains are not conservativereplacements for those of the L-amino acid being replaced. This isbecause of the difference in chirality and hence side-chain orientation,which may result in the accessing of a previously unexplored region ofthe binding site of the target which has moieties of different charge,hydrophobicity, steric requirements, etc., than that serviced by theside chain of the replaced L-amino acid.

Modifications to Improve Pharmacokinetic or Pharmacodynamic Properties

It is also understood that use of the binding moieties of the inventionin a particular application may necessitate modifications of the peptideor formulations of the peptide to improve pharmacokinetic andpharmacodynamic behavior. It is expected that the properties of thepeptide may be changed by attachment of moieties anticipated to bringabout the desired physical or chemical properties. Such moietiesaffecting the pharmacokinetic and pharmacodynamic behavior may beappended to the peptide using acids or amines, via amide bonds or ureabonds, respectively, to the N- or C-terminus of the peptide, or to thependant amino group of a suitably located lysine or lysine derivative,diaminopropionic acid, ornithine, or other amino acid in the peptidethat possesses a pendant amine group or a pendant alkoxyamino orhydrazine group. The moieties introduced may be groups that arehydrophilic, basic, or nonpolar alkyl or aromatic groups depending onthe peptide of interest and the extant requirements for modification ofits properties.

Glycosylation of Amino Acid Residues

Yet another modification within the scope of the invention is to employglycosylated amino acid residues (e.g. serine, threonine or asparagineresidues), singly or in combination in either the binding moiety or thelinker moiety or both. Glycosylation, which may be carried out usingstandard conditions, may be used to enhance solubility, alterpharmacokinetics and pharmacodynamics or to enhance binding via aspecific or non-specific interaction involving the glycosidic moiety. Inanother approach glycosylated amino acids such asO-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl) serine orthe analogous threonine derivative (either the D- or L-amino acids) maybe incorporated into the peptide during manual or automated solid phasepeptide synthesis, or in manual or automated solution phase peptidesynthesis. Similarly D- orL-N^(γ)-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl)-asparaginecan be employed. The use of amino acids glycosylated on a pendantoxygen, nitrogen or sulfur function by the agency of suitablyfunctionalized and activated carbohydrate moieties that can be employedin glycosylation is anticipated. Such carbohydrate functions could bemonosaccharides, disaccharides or even larger assemblies ofoligosaccharides (Kihlberg, January (2000) Glycopeptide synthesis. In:Fmoc Solid Phase Peptide Synthesis—A Practical Approach (Chan, W. C. andWhite, P. D. Eds) Oxford University Press, New York, N.Y. Chap. 8, pp195-213).

Also anticipated is the appendage of carbohydrate functions to aminoacids by means other than glycosylation via activation of a leavinggroup at the anomeric carbon. Linkage of the amino acid to the glycosideis not limited to the formation of a bond to the anomeric carbon of thecarbohydrate function. Instead, linkage of the carbohydrate moiety tothe amino acid could be through any suitable, sufficiently reactiveoxygen atom, nitrogen atom, carbon atom or other pendant atom of thecarbohydrate function via methods employed for formation ofC-heteroatom, C—C or heteroatom-heteroatom (examples are S—S, O—N, N—N,P—O, P—N) bonds known in the art.

Formation of Salts

It is also within the scope of the invention to form different saltsthat may increase the water solubility or the ease of formulation ofthese peptides. These may include, but are not restricted to,N-methylglucamine (meglumine), acetate, oxalates, ascorbates etc.

Structural Modifications which Retain Structural Features

Yet another modification within the scope of the invention is truncationof cyclic polypeptides. The cyclic nature of many polypeptides of theinvention limits the conformational space available to the peptidesequence, particularly within the cycle. Therefore truncation of thepeptide by one or more residues distal or even proximal to the cycle, ateither the N-terminal or C-terminal region may provide truncatedpeptides with similar or improved biological activity. A unique sequenceof amino acids, even as small as three amino acids, which is responsiblefor the binding activity, may be identified, as noted for RGD peptides.See e.g., E. F. Plow et al., Blood (1987), 70(1), 110-5; A. Oldberg etal., Journal of Biological Chemistry (1988), 263(36), 19433-19436; R.Taub et al., Journal of Biological Chemistry (1989 Jan. 5), 264(1),259-65; A. Andrieux et al., Journal of Biological Chemistry (1989 Jun.5), 264(16), 9258-65; and U.S. Pat. Nos. 5,773,412 and 5,759,996, eachof which is incorporated herein by reference in its entirety.

It has also been shown in the literature that large peptide cycles canbe substantially shortened, eliminating extraneous amino acids, butsubstantially including the critical binding residues. See U.S. Pat. No.5,556,939, which is incorporated herein by reference in its entirety.Shortened cyclic peptides can be formed using disulfide bonds or amidebonds of suitably located carboxylic acid groups and amino groups.

Furthermore, D-amino acids can be added to the peptide sequence tostabilize turn features (especially in the case of glycine). In anotherapproach alpha, beta, gamma or delta dipeptide or turn mimics (such asα, β, γ, or δ turn mimics) some of which are shown in structures 1, 2and 3, below, can be employed to mimic structural motifs and turnfeatures in a peptide and simultaneously provide stability fromproteolysis and enhance other properties such as, for example,conformational stability and solubility (structure 1: Hart et al., J.Org. Chem., 64, 2998-2999(1999); structure 2: Hanessian et al.,“Synthesis of a Versatile Peptidomimetic Scaffold” in Methods inMolecular Medicine, Vol. 23: Peptidomimetics Protocols, W. M. KazmierskiEd. (Humana Press Inc. Totowa N.J. 1999), Chapter 10, pp. 161-174;structure 3: WO 01/16135.

Substitution of Disulfide Mimetics

Also included within the scope of the invention is the substitution ofdisulfide mimetics for disulfide bonds within the binding polypeptidesof the invention. For disulfide-containing peptides of the invention,the disulfide bonds might need to be replaced to avoid certaindifficulties that are sometimes posed by the presence of a disulfidebond. For example, when generating ^(99m)Tc (or otherradionuclide)-based radiopharmaceuticals or certain other constructswith binding peptides of the invention, the presence of the disulfidebond can be a significant problem. The integrity of the disulfide bondis difficult to maintain during procedures designed to incorporate^(99m)Tc via routes that are reliant upon the reduction of pertechnetateion and subsequent incorporation of the reduced Tc species intosubstances bearing Tc-compatible chelating groups. This is because thedisulfide bond is rather easily reduced by the reducing agents commonlyused in kits devised for one-step preparation of radiopharmaceuticals.Therefore, the ease with which the disulfide bond can be reduced duringTc chelation may require substitution with mimetics of the disulfidebonds. Accordingly, another modification within the scope of theinvention is to substitute the disulfide moiety with mimetics, utilizingthe methods disclosed herein or known to those skilled in the art, whileretaining the activity and other desired properties of the bindingpolypeptides used in the invention:

Oxime Linker

The oxime moiety has been employed as a linker by investigators in anumber of contexts. Of the most interest is the work by Wahl, F andMutter, M, Tetrahedron Lett. (1996) 37, 6861-6864). The amino acidscontaining an aminoalcohol function (4), and containing an alkoxyaminofunction (5), are incorporated into the peptide chain, not necessarilyat the end of the peptide chain. After formation of the peptide, thesidechain protecting groups are removed. The aldehyde group is unmaskedand an oxime linkage is formed.

Lanthionine Linker

Lanthionines are cyclic sulfides, wherein the disulfide linkage (S—S) isreplaced by a (C—S) linkage. Thus the lability to reduction is far lowerand this linkage should be stable to stannous chloride. Lanthionines maybe prepared by a number of methods.

Preparation of Lanthionines Using Bromoacetylated Peptides

Lanthionines are readily prepared using known methods. See, for example,Robey et al. (Robey, F. A. and Fields, R. L. Anal. Biochem. (1989) 177,373-377) and Inman, et al. (Inman, J. K.; Highet, P. F.; Kolodny, N.;and Robey, F. A. Bioconjugate Chem. (1991) 2, 458-463; Ploinsky, A.Cooney, M. C. Toy-Palmer, A. Osapay, G. and Goodman, M. J. Med. Chem.(1992) 35, 4185-4194; Mayer, J. P.; Zhang, J.; and Liu, C. F. in: Tam,J. P. and Kaumaya, P. T. P. (eds), “Peptides, Frontiers of PeptideScience,” Proceedings of the 15^(th) American Peptide Symposium, June14-19 Nashville, Tenn. Klumer Academic Pub. Boston. pp 291-292;. Wakao,Norihiro; Hino, Yoichi; Ishikawa, Ryuichi. Jpn. Kokai Tokkyo Koho(1995), 7 pp. JP 07300452 A2 19951114 Heisei; JP 95-49692 19950309; JP94-41458 19940311 have published in this area. Preparation of peptidesusing Boc automated peptide synthesis followed by coupling the peptideterminus with bromoacetic acid gives bromoacetylated peptides in goodyield. Cleavage and deprotection of the peptides is accomplished usingHF/anisole. If the peptide contains a cysteine group its reactivity canbe controlled with low pH. If the pH of the medium is raised to 6-7,then either polymerization or cyclization of the peptide takes place.Polymerization is favored at high (100 mg/mL) concentration, whereascyclization is favored at lower concentrations (1 mg/mL), e.g., inScheme 1 below, 6 cyclizes to 7.

Inman et al. demonstrated the use ofN^(α)-(Boc)-N^(ε)-[N-(bromoacetyl)-β-alanyl]-L-lysine as a carrier ofthe bromoacetyl group that could be employed in Boc peptide synthesisthus allowing placement of a bromoacetyl bearing moiety anywhere in asequence. In preliminary experiments they found that peptides with 4-6amino acids separating the bromoacetyl-lysine derivative from a cysteinetend to cyclize, indicating the potential utility of this strategy.

Preparation of Lanthionines via Cysteine Thiol Addition to Acrylamides

Several variants of this strategy may be implemented. Resin-bound serinecan be employed to prepare the lanthionine ring on resin either using abromination-dehydrobromination-thiol addition sequence or by dehydrationwith disuccinimidyl carbonate followed by thiol addition. Ploinsky etal., M. J. Med. Chem., 35:4185-4194 (1992); Mayer et al., “Peptides,Frontiers of Peptide Science”, in Proceedings of the 15^(th) AmericanPeptide Symposium, Tam & Kaumaya (eds), Jun. 14-19, 1995, Nashville,Tenn. (Klumer Academic Pub. Boston) pp. 291-292. Conjugate addition ofthiols to acrylamides has also been amply demonstrated and a referenceto the addition of 2-mercaptoethanol to acrylamide is provided. Wakao etal., Jpn. Kokai Tokkyo Koho, JP 07300452 A2 (1995).

Diaryl Ether or Diarylamine Linkage

Diaryl Ether Linkage from Intramolecular Cyclization of Aryl BoronicAcids and Tyrosine

The reaction of arylboronic acids with phenols, amines and heterocyclicamines in the presence of cupric acetate, in air, at ambienttemperature, in dichloromethane using either pyridine or triethylamineas a base to provide unsymmetrical diaryl ethers and the related aminesin good yields (as high as 98%) has been reported. See, Evans et al.,Tetrahedron Lett., 39:2937-2940 (1998); Chan et al., Tetrahedron Lett.,39:2933-2936 (1998); Lam et al., Tetrahedron Lett., 39:2941-2944 (1998).In the case of N-protected tyrosine derivatives as the phenol componentthe yields were also as high as 98%. This demonstrates that amino acidamides (peptides) are expected to be stable to the transformation andthat yields are high. Precedent for an intramolecular reaction exists inview of the facile intramolecular cyclizations of peptides to lactams,intramolecular biaryl ether formation based on the S_(N)Ar reaction andthe generality of intramolecular cyclization reactions under highdilution conditions or on resin, wherein the pseudo-dilution effectmimics high dilution conditions.

Formation of Cyclic Peptides with a Lactam Linkage via IntramolecularNative Chemical Ligation

Another approach that may be employed involves intramolecularcyclization of suitably located vicinal amino mercaptan functions(usually derived from placement of a cysteine at a terminus of thelinear sequence or tethered to the sequence via a side-chain nitrogen ofa lysine, for example) and aldehyde functions to provide thiazolidineswhich result in the formation of a bicyclic peptide, one ring of whichis that formed by the residues in the main chain, and the second ringbeing the thiazolidine ring. Scheme 2, above, provides an example. Therequired aldehyde function can be generated by sodium metaperiodatecleavage of a suitably located vicinal aminoalcohol function, which canbe present as an unprotected serine tethered to the chain by appendageto a side chain amino group of a lysine moiety. In some cases, therequired aldehyde function is generated by unmasking of a protectedaldehyde derivative at the C-terminus or the N-terminus of the chain. Anexample of this strategy is found in: Botti, P.; Pallin, T. D. and Tam,J. P. J. Am. Chem. Soc. 1996, 118, 10018-10034.

Lactams Based on Intramolecular Cyclization of Pendant Amino Groups withCarboxyl Groups on Resin

Macrocyclic peptides can be prepared by lactam formation by either headto tail or by pendant group cyclization. The basic strategy is toprepare a fully protected peptide wherein it is possible to removeselectively an amine protecting group and a carboxy protecting group.Orthogonal protecting schemes have been developed. Of those that havebeen developed, the allyl, trityl and Dde methods have been employedmost. See, Mellor et al., “Synthesis of Modified Peptides,” in FmocSolid Phase Synthesis: A Practical Approach, White and Chan (eds)([Oxford University Press, New York, 2000]), Chapt. 6, pp. 169-178. TheDde approach is of interest because it utilizes similar protectinggroups for both the carboxylic acid function (Dmab ester) and the aminogroup (Dde group). Both are removed with 2-10% hydrazine in DMF atambient temperature. Alternatively, the Dde can be used for the aminogroup and the allyl group can be used for the carboxyl.

A lactam function, available by intramolecular coupling via standardpeptide coupling reagents (such as HATU, PyBOP etc), could act as asurrogate for the disulfide bond. The Dde/Dmab approach is shown inScheme 3a, below.

Thus, a linear sequence containing, for example, the Dde-protectedlysine and Dmab ester may be prepared on a Tentagel-based Rink amideresin at low load (˜0.1-0.2 mmol/g). Deprotection of both functions withhydrazine is then followed by on-resin cyclization to give the desiredproducts.

In the allyl approach, shown in Scheme 3b, the pendant carboxyl which isto undergo cyclization is protected as an allyl ester and the pendantamino group is protected as an alloc group. On resin, both areselectively unmasked by treatment with palladium tris-triphenylphosphinein the presence of N-methylmorpholine and acetic acid in DMF. Residualpalladium salts are removed using sodium diethyldithiocarbamate in thepresence of DIEA in DMF, followed by subsequent washings with DMF. Thelactam ring is then formed employing HATU/HOAt in the presence ofN-methylmorpholine. Other coupling agents can be employed as describedabove. The processing of the peptide is then carried out as describedabove to provide the desired peptide lactam.

Subsequently cleavage from resin and purification may also be carriedout. For functionalization of the N-terminus of the peptide, it isunderstood that amino acids, such astrans-4-(iV-Dde)methylaminocyclohexane carboxylic acid,trans-4-(iV-Dde)methylaminobenzoic acid, or their alloc congeners couldbe employed. Yet another approach is to employ the safety catch methodto intramolecular lactam formation during cleavage from the resin.

Cyclic Peptides Based on Olefin Metathesis

The Grubbs reaction (Scheme 4, below) involves themetathesis/cyclization of olefin bonds and is illustrated as shownbelow. See, Schuster et al., Angewandte. Chem. Int. Edn Engl.,36:2036-2056 (1997); Miller et al., J. Am. Chem. Soc., 118:9606-9614(1996).

It is readily seen that, if the starting material is a diolefin (16),the resulting product will be cyclic compound 17. The reaction has infact been applied to creation of cycles from olefin-functionalizedpeptides. See, e.g., Pernerstorfer et al., Chem. Commun., 20:1949-50(1997); Covalent capture and stabilization of cylindrical β-sheetpeptide assemblies, Clark et al., Chem. Eur. J., 5(2):782-792 (1999);Highly efficient synthesis of covalently cross-linked peptide helices byring-closing metathesis, Blackwell et al., Angew. Chem., Int. Ed.,37(23):3281-3284 (1998); Synthesis of novel cyclic protease inhibitorsusing Grubbs olefin metathesis, Ripka et al., Med. Chem. Lett.,8(4):357-360 (1998); Application of Ring-Closing Metathesis to theSynthesis of Rigidified Amino Acids and Peptides, Miller et al., J. Am.Chem. Soc., 118(40):9606-9614 (1996); Supramolecular Design by CovalentCapture, Design of a Peptide Cylinder via Hydrogen-Bond-PromotedIntermolecular Olefin Metathesis, Clark et al., J. Am. Chem. Soc.,117(49):12364-12365 (1995); Synthesis of Conformationally RestrictedAmino Acids and Peptides Employing Olefin Metathesis, Miller et al., J.Am. Chem. Soc., 117(21):5855-5856 (1995). One can prepare eitherC-allylated amino acids or possibly N-allylated amino acids and employthem in this reaction in order to prepare carba-bridged cyclic peptidesas surrogates for disulfide bond containing peptides.

One may also prepare novel compounds with olefinic groups.Functionalization of the tyrosine hydroxyl with an olefin-containingtether is one option. The lysine ε-amino group may be another optionwith appendage of the olefin-containing unit as part of an acylatingmoiety, for example. If instead the lysine side chain amino group isalkylated with an olefin containing tether, it can still function as apoint of attachment for a reporter as well. The use of 5-pentenoic acidas an acylating agent for the lysine, ornithine, or diaminopropionicside chain amino groups is another possibility. The length of theolefin-containing tether can also be varied in order to explorestructure activity relationships.

Manipulation of Peptide Sequences

Other modifications within the scope of the invention includemanipulations of peptide sequences which can be expected to yieldpeptides with similar or improved biological properties. These includeamino acid translocations (swapping amino acids in the sequence), use ofretro-inverso peptides in place of the original sequence or a modifiedoriginal sequence, peptoids, retro-inverso peptoid sequences, andsynthetic peptides. Structures wherein specific residues are peptoidinstead of peptidic, which result in hybrid molecules, neithercompletely peptidic nor completely peptoid, are contemplated as well.

The peptides or conjugates of the invention may include one or moreprotecting groups on any appropriate amino or carboxylic groups or otherappropriate functional groups. Unless otherwise noted, the term“protecting group” refers to a protective group adapted to preserve thecharacteristic chemical function of the functional group to which it isbound. For example, protecting groups may be used to preserve amino orcarboxyl functions and may include, for example, Fmoc, benzyl,benzyloxycarbonyl or alkyl esters or other groups commonly intended forprotection of such functions. Additional protecting groups are disclosedin, for example, T. W. Green, Protective Groups in Organic Synthesis(Wiley, N.Y. 1981).

In appropriate circumstances the peptides or conjugates of the inventionmay also include a “deactivating group”, which refers to a chemicalgroup that is able to chemically react with, for example, the N terminal(—NH2) or C-terminal (—COOH) group of the peptide unit, transforming itthrough a chemical reaction, into a suitable derivative thereof thatmaintains the specificity of the corresponding peptide moiety towardfibrin, but is unable to chemically react with, respectively, a carboxylor an amino functionality on a different moiety, and thus may not beinvolved in carboxamido reactions. Such groups may include acetyl (alsoreferred to as CH3(CO)— or Ac), amino groups and derivatives thereofsuch as, for example, —NH2, —NH(CH3), H2NOC—CH2-NH—.

Multimeric constructs including fibrin-binding peptides of the inventionmay be prepared using known linkers and techniques, such as, forexample, those set forth in co-pending U.S.S.N. (US 2005/0147555),incorporated herein by reference in its entirety. In a preferredembodiment homodimers including fibrin-binding peptides may be prepared.Such dimeric compounds have increased avidity and thus exhibit betterbinding to fibrin.

In the practice of the present invention, a determination of theaffinity of the fibrin-binding moiety for fibrin relative to fibrinogenis a useful measure, and is referred to as specificity for fibrin.Standard assays for quantitating binding and determining affinityinclude equilibrium dialysis, equilibrium binding, gel filtration, orthe monitoring of numerous spectroscopic changes (such as a change influorescence polarization) that may result from the interaction of thebinding moiety and its target. These techniques measure theconcentration of bound and free ligand as a function of ligand (orprotein) concentration. The concentration of bound polypeptide ([Bound])is related to the concentration of free polypeptide ([Free]) and theconcentration of binding sites for the polypeptide, i.e., on fibrin,(N), as described in the following equation:[Bound]=N×[Free]/((1/K _(a))+[Free]).

A solution of the data to this equation yields the association constant,K_(a), a quantitative measure of the binding affinity. The associationconstant, K_(a) is the reciprocal of the dissociation constant, K_(D).The K_(D) is more frequently reported in measurements of affinity. Apeptide having a K_(D) 1.5 times higher for fibrinogen than for fibrinwould be considered low-specificity fibrin binder. A peptide having aK_(D) 10 times greater for fibrinogen than fibrin would be amoderate-specificity fibrin binder, and a peptide having a K_(D) 100times or more greater for fibrinogen than for fibrin would be termedhighly specific for fibrin. Preferably the peptides and agents of thepresent invention have a K_(D) at least 1.5 times higher for fibrinogenthan for fibrin, more preferably at least 10 times higher, even morepreferably at least 100 times, and most preferably at least 1000 timeshigher. Preferred fibrin binding polypeptides have a K_(D) for fibrin inthe range of 1 nanomolar (nM) to 100 micromolar (μM) and includes K_(D)values of at least 10 nM, at least 20 nM, at least 40 nM, at least 60nM, at least 80 nM, at least 1 μM, at least 5 μM, at least 10 μM, atleast 20 μM, at least 40 μM, at least 60 μM, and at least 80 μM.

Where fibrin binding moieties are employed as imaging agents, otheraspects of binding specificity may become more important: Imaging agentsoperate in a dynamic system in that binding of the imaging agent to thetarget is not in a stable equilibrium state throughout the imagingprocedure. For example, when the imaging agent is initially injected,the concentration of imaging agent and of agent-target complex rapidlyincreases. Shortly after injection, however, the circulating (free)imaging agent starts to clear through the kidneys or liver, and theplasma concentration of imaging agent begins to drop. This drop in theconcentration of free imaging agent in the plasma eventually causes theagent-target complex to dissociate. The usefulness of an imaging agentdepends on the difference in rate of agent-target dissociation relativeto the clearing rate of the agent. Ideally, the dissociation rate willbe slow compared to the clearing rate, resulting in a long imaging timeduring which there is a high concentration of agent-target complex and alow concentration of free imaging agent (background signal) in theplasma. The dissociation rate of the complex is controlled by thedissociation rate constant, k_(o)ff. Because higher values of k_(o)ffcorrespond to faster dissociation rates, it is preferable to obtainbinding peptides which have a low k_(o)ff for use as imaging agents.

The fibrin-binding moieties according to this invention will beextremely useful for detection and/or imaging of fibrin in vitro or invivo, and particularly for detection and/or imaging of fibrin clots andpathological angiogenic processes. Any suitable method of assaying orimaging fibrin may be employed.

For detection of fibrin or fibrin-derived polypeptides in solution, abinding moiety according to the invention can be detectably labeled,e.g., fluorescently labeled, radiolabeled or enzymatically labeled, thencontacted with the solution, and thereafter formation of a complexbetween the binding moiety and the fibrin target can be detected. As anexample, a fluorescently labeled fibrin binding peptide may be used forin vitro fibrin detection assays, wherein the peptide is added to asolution to be tested for fibrin under conditions allowing binding tooccur. The complex between the fluorescently labeled fibrin-bindingpeptide and fibrin can be detected and quantified by measuring theincreased fluorescence polarization arising from the fibrin-boundpeptide relative to that of the free peptide.

Alternatively, a sandwich-type ELISA assay may be used, wherein a fibrinbinding moiety is immobilized on a solid support such as a plastic tubeor well, then the solution suspected of containing fibrin or afibrin-derived polypeptide is contacted with the immobilized bindingmoiety, non-binding materials are washed away, and complexed polypeptideis detected using a suitable detection reagent, such as a monoclonalantibody recognizing fibrin. The monoclonal antibody is detectable byconventional means known in the art, including being detectably labeled,e.g., radiolabeled, conjugated with an enzyme such as horseradishperoxidase and the like, or fluorescently labeled.

For detection or purification of soluble fibrin or fibrin-derivedpolypeptides in or from a solution, a binding moiety of the inventioncan be immobilized on a solid substrate such as a chromatographicsupport or other porous material, then the immobilized binder can beloaded or contacted with the solution under conditions suitable forformation of a binding moiety/fibrin complex. The non-binding portion ofthe solution can be removed and the complex may be detected, e.g., usingan anti-fibrin or anti-binding moiety antibody, or the fibrin target maybe released from the binding moiety at appropriate elution conditions.

A particularly preferred use for the polypeptides according to thepresent invention is for creating visually readable images of thrombiand pathologic angiogenic processes, to aid in the diagnosis, monitoringand treatment of such disorders. The fibrin binding polypeptidesdisclosed herein may be converted to imaging reagents for detectingthrombi by conjugating the polypeptides with a label appropriate fordiagnostic detection. Such labels, referred to, for example, as adetectable label or a diagnostically effective any moiety, include anymoiety detectable by imaging procedures, that is to say any moiety ableto provide, to improve or, in any way, to advantageously modify thesignal detected by a diagnostic imaging technique including, forinstance, magnetic resonance imaging (MRI), radioimaging, X-ray imaging,light imaging, ultrasound imaging, thus enabling the registration ofdiagnostically useful, preferably contrasted, images when used inassociation with the said techniques. Examples of detectable labels ordiagnostically effective moieties according to the invention include,for instance, chelated gamma ray or positron emitting radionuclides;paramagnetic metal ions in the form of chelated or polychelatedcomplexes, X-ray absorbing agents including atoms having atomic numberhigher than 20; an ultrasound contrast agent, including for examplegas-filled microvesicle, a dye molecule; a fluorescent molecule; aphosphorescent molecule; a molecule absorbing in the UV spectrum; aquantum dot; a molecule capable of absorption within near or farinfrared radiations and, in general, all the moieties which generate adetectable substance.

Preferably, a peptide of the invention exhibiting a strong ability tobind fibrin is conjugated or linked (directly or via a linker) to alabel appropriate for the detection methodology to be employed. Forexample, the fibrin binder may be conjugated with a paramagnetic chelatesuitable for magnetic resonance imaging (MRI), with a radiolabelsuitable for x-ray imaging, with an ultrasound microsphere or liposomesuitable for ultrasound detection, or with an optical imaging dye.

Alternatively, a peptide of the invention exhibiting a strong ability tobind fibrin is conjugated or linked (directly or via a linker) to atherapeutic agent. Such compounds are useful for treatment oralleviation of diseases associated with fibrin.

Additional modifications within the scope of the invention includeintroduction of linkers or spacers between the targeting sequence of thefibrin binding peptide and the detectable label or therapeutic agent.Use of such linkers/spacers may improve the relevant properties of thebinding peptide (e.g., improve the binding ability, increase serumstability, adjust hydrophobicity or hydrophilicity, providing improvedpharmacokinetic and pharmacodynamic properties, etc.). Indeed, use of anappropriate linker and/or spacer may provide an optimal distance betweenthe fibrin-binding peptide and the detectable label or therapeuticagent, which may in turn improve the targeting capability of thecompounds of the invention. These linkers may include, but are notrestricted to, substituted or unsubstituted, saturated or unsaturated,straight or branched alkyl chains, derivatized or underivatizedpolyethylene glycol, polyoxyethylene or polyvinylpyridine chains, one ormore amino acids (and preferably 3 or more amino acids and mostpreferably at least 4 or 6 amino acids, peptides from straight, branchedor cyclic amino acids, sugars, or aliphatic or aromatic spacers commonin the art, substituted or unsubstituted polyamide chains; derivatizedor underivatized polyamine, polyester, polyethylenimine, polyacrylate,poly(vinylalcohol), polyglycerol, or oligosaccharide (e.g., dextran)chains; glycosylated amino acid residues, alternating block copolymers;malonic, succinic, glutaric, adipic and pimelic acids; caproic acid;simple diamines and dialcohols; any of the other linkers disclosedherein as well as any other simple polymeric linker known in the art,for instance as described in WO 98/18497 and WO 98/18496.

Furthermore, linkers that are combinations of the moieties describedabove, can also be employed to confer special advantage to theproperties of the peptide. The linker may be a linear or branched and ispreferably at least a divalent linking moiety. A “divalent linkingmoiety is a chain including two functional groups allowing for itsconjugation with a peptide on one side and a detectable label ortherapeutic agent on another. Preferably the divalent linking moietypermits conjugation with the terminal amino or carboxyl groups of thepeptide and an appropriate functional group on the detectable label ortherapeutic agent. A “functional group” refers to specific groups ofatoms within molecules or moieties that are responsible for thecharacteristic chemical reaction of those molecules or moieties and mayinclude, for example, the —NH2 or —COOH groups of peptides, as well asother active groups of detactable labels or therapeutic agents, such, asfor example, amino, thiol or carboxyl groups.

The linker may also be a polyfunctional linking moiety or polyfunctionallinker, which refers to a linear or branched chain including at least 3functional groups, one of them connecting the linking moiety with thepeptide, and the remainder connecting the linking moiety with at leasttwo detectable labels and/or therapeutic agents. Such polyfunctionallinking moieties may include, for example, N-branched Lysine systems(see, f. i., Veprek, P et al., J. Pept. Sci. 5, 5 (1999); 5, 203 (1999),polycarboxylic compounds and suitable derivative thereof in which thecarboxylic group(s) are in a suitably activated or protected form,polyaminated compounds and suitable derivative thereof in which theamino group(s) are in a suitably activated or protected form, and aminoacids and poly-amino acids such as polyornithine, polyarginine,polyglutamic acid, polyaspartic acid.

Lipid molecules with linkers may be attached to allow formulation ofultrasound bubbles, liposomes or other aggregation based constructs.Such constructs could be employed as agents for targeting and deliveryof a diagnostic reporter, a therapeutic agent (e.g., a chemical“warhead” for therapy) or a combination of these.

In the present invention an especially preferred linker is GGGK.Additionally, the linkers GSAGSK (SEQ ID NO. 137) and GAGSGK (SEQ ID NO.138) are also preferred.

In general, the technique of using a detectably labeled fibrin bindingmoiety is based on the premise that the label generates a signal that isdetectable outside the patient's body. When the detectably labeledfibrin binding moiety is administered to the patient suspected of havinga fibrin related disorder (such as a thrombus or pathological angiogenicprocess), the high affinity of the fibrin binding moiety for fibrincauses the binding moiety to bind to fibrin and accumulate label at thesite of interest. The signal generated by the labeled peptide isdetected by a scanning device which will vary according to the type oflabel used, and the signal is then converted to an image of the area.

Magnetic Resonance Imaging

The fibrin binding moieties of the present invention may advantageouslybe conjugated with a MRI detectable moiety, such as, for example, aparamagnetic metal chelator or iron particles (such as superparamagneticFeO particles) in order to form a contrast agent for use in MRI.Preferred paramagnetic metal ions have atomic numbers 21-31, 39, 42, 43,44, 49 or 57-83. This includes ions of the transition metal orlanthanide series which have one, and more preferably five or more,unpaired electrons and a magnetic moment of at least 1.7 Bohr magneton.The preferred paramagnetic metal is selected from the group consistingof Fe(2+), Fe(3+), Cu(2+), Ni(2+), Rh(2+), Co(2+), Cr(3+), Gd(3+),Eu(3+), Dy(3+), Tb(3+), Pm(3+), Nd(3+), Tm(3+), Ce(3+), Y(3+), Ho(3+),Er(3+), La(3+), Yb(3+), Mn(3+), Mn(2+). Gd(3+) (also referred to asGd(III)) is particularly preferred for MRI due to its high relaxivityand low toxicity, and the availability of only one biologicallyaccessible oxidation state. Gd(III) chelates have been used for clinicaland radiologic MR applications since 1988, and approximately 30% of MRexams currently employ a gadolinium-based contrast agent. Additionally,fibrin-binding moieties of the present invention also can be conjugatedwith other MRI detectable moieties, such as, for example, one or moresuperparamagnetic particles.

The practitioner will select a metal according to dose required todetect a thrombus and considering other factors such as toxicity of themetal to the subject. See, Tweedle et al., Magnetic Resonance Imaging(2nd ed.), vol. 1, Partain et al., eds. (W. B. Saunders Co. 1988), pp.796-7. Generally, the desired dose for an individual metal will beproportional to its relaxivity, modified by the biodistribution,pharmacokinetics and metabolism of the metal. The trivalent cation, Gd³⁺is particularly preferred for MRI contrast agents, due to its highrelaxivity and low toxicity, with the further advantage that it existsin only one biologically accessible oxidation state, which minimizesundesired metabolization of the metal by a patient. Another useful metalis Cr³⁺, which is relatively inexpensive.

A chelator (also called a chelating ligand or chelating agent) is achemical moiety, agent, compound or molecule having one or more polargroups that act as a ligand for, and complex with, a paramagnetic metal.Suitable chelators are known in the art and include acids with methylenephosphonic acid groups, methylene carbohydroxamine acid groups,carboxyethylidene groups, or carboxymethylene groups. In a preferredembodiment the chelators includes cyclic or linear polyaminopolycarboxylic or polyphosphonic acids and contains at least one amino,thiol or carboxyl group. Examples of chelators include, but are notlimited to, a polyaminopolycarboxylic acid and the derivatives thereof,comprising, for example, diethylenetriamine pentaacetic acid (DTPA) andderivatives thereof such as benzo-DTPA, dibenzo-DTPA, phenyl-DTPA,diphenyl-DTPA, benzyl-DTPA, dibenzyl DTPA;N,N-bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]amino]ethyl]-glycine(DTPA-BMA);N-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl)]-N-[2-[bis(carboxymethyl)amino]ethylglycine(EOB-DTPA);4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oicacid (BOPTA);N,N-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamicacid 1-(1,1-dimethylethyl)esterN,N-bis[2-[bis(carboxymethyl)amino]ethyl]L-glutamic acid (DTPA-GLU);DTPA conjugated with Lys (DTPA-Lys); ethylenediaminetetraacetic acid(EDTA); 1,4,7,10-teraazacyclododecane 1,4,7,-triacetic acid (DO3A) andderivatives thereof including, for example,[10-(2-hydroxypropyl)-1,4,7,10-teraazacyclododecane 1,4,7,-triaceticacid (HPDO3A); 1,4,7-triazacyclononane N,N′,N″-triacetic acid (NOTA);2-methyl-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid(MCTA);6-[bis(carboxymethyl)amino]tetrahydro-6-methyl-1H-1,4-diazepine-1,4(5H)-diaceticacid (AAZTA) provided by WO03008390 application, incorporated herein byreference, and derivatives thereof;1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA) andderivatives thereof, including for instance, benzo-DOTA, dibenzo-DOTA,(α,α′,α″,α′″)-tetramethyl-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraaceticacid (DOTMA); or1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA);or corresponding compounds wherein one or more of the carboxylic groupsis replaced by a phosphonic and/or phosphinic group, including, forinstance, N,N′-bis-(pyridoxal-5-phosphate) ethylenediamine-N,N′-diaceticacid (DPDP); ethylenedinitrilotetrakis(methylphosphonic) acid (EDTP),1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methylenephosphonic)acid (DOTP), the phosphonoalkyl-polyaza macrocyclic compounds forinstance disclosed in U.S. Pat. No. 5,362,476 and U.S. Pat. No.5,409,689 and the linear phosphonoalkyl derivatives disclosed in U.S.Pat. No. 6,509,324; or of macrocyclic chelants such as texaphirines,porphyrins, phthalocyanines.

Additional chelating ligands are ethylenebis-(2-hydroxy-phenylglycine)(EHPG), and derivatives thereof, including 5-Cl-EHPG, 5Br-EHPG,5-Me-EHPG, 5t-Bu-EHPG, and 5sec-Bu-EHPG; bis-2(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivativesthereof, derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl) aminomethylbenzene (MECAM).Examples of representative chelators and chelating groups contemplatedby the present invention are described in, for example, WO 98/18496, WO86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO 97/36619,PCT/US98/01473, PCT/US98/20182, and U.S. Pat. No. 4,899,755, all ofwhich are hereby incorporated by reference.

Preferred ligands according to the present invention are set forth inFIGS. 6 a to 6 c, together with suitable bibliographic referencesconcerning their preparation.

Particularly preferred are: DTPA, DTPA-GLU, DTPA-Lys, DOTA, AAZTA, andthe following AAZTA derivatives:

In accordance with the present invention, the chelator of the MRIcontrast agent is coupled to the fibrin binding moiety directly or via alinker. The positioning of the chelator should be selected so as not tointerfere with the binding affinity or specificity of the fibrin bindingmoiety. Preferably, the chelate will be appended either to the Nterminus or the C terminus, however the chelate may also be attachedanywhere within the sequence. In a preferred embodiment, a chelatorhaving a free central carboxylic acid group (e.g.,DTPA-Asp(β.-COOH)-OtBu) makes it easy to attach at the N-terminus of thepeptide by formation of an amide bond. The chelate could also beattached at the C-terminus with the aid of a linker. Alternatively,isothiocyanate conjugation chemistry could be employed as a way oflinking the appropriate isothiocyanto group bearing DTPA to a free aminogroup anywhere within the peptide sequence.

In general, the fibrin binding moiety can be bound directly orcovalently to the metal chelator (or other detectable label), or it maybe coupled or conjugated to the metal chelator using a linker, which maybe, without limitation, amide, urea, acetal, ketal, double ester,carbonyl, carbamate, thiourea, sulfone, thioester, ester, ether,disulfide, lactone, imine, phosphoryl, or phosphodiester linkages;substituted or unsubstituted saturated or unsaturated alkyl chains;linear, branched, or cyclic amino acid chains of a single amino acid ordifferent amino acids (e.g., extensions of the N- or C-terminus of thefibrin binding moiety); derivatized or underivatized polyethyleneglycol, polyoxyethylene, or polyvinylpyridine chains; substituted orunsubstituted polyamide chains; derivatized or underivatized polyamine,polyester, polyethylenimine, polyacrylate, poly(vinyl alcohol),polyglycerol, or oligosaccharide (e.g., dextran) chains; alternatingblock copolymers; malonic, succinic, glutaric, adipic and pimelic acids;caproic acid; simple diamines and dialcohols; and other simple polymericlinkers known in the art (see, e.g., WO 98/18497, WO 98/18496) or otherlinkers discussed herein. Preferably the molecular weight of the linkercan be tightly controlled. The molecular weights can range in size fromless than 100 to greater than 1000. Preferably the molecular weight ofthe linker is less than 100. In addition, it may be desirable to utilizea linker that is biodegradable in vivo to provide efficient routes ofexcretion for the imaging reagents of the present invention. Dependingon their location within the linker, such biodegradable functionalitiescan include ester, double ester, amide, phosphoester, ether, acetal, andketal functionalities.

In general, known methods can be used to couple the metal chelate andthe fibrin binding moiety using such linkers. See, e.g., WO 95/28967, WO98/18496, WO 98/18497 and discussion therein. The fibrin binding moietycan be linked through its N- or C-terminus via an amide bond, forexample, to a metal coordinating backbone nitrogen of a metal chelate orto an acetate arm of the metal chelate itself. The present inventioncontemplates linking of the chelate on any position, provided the metalchelate retains the ability to bind the metal tightly in order tominimize toxicity. Similarly, the fibrin binding moiety may be modifiedor elongated in order to generate a locus for attachment to a metalchelate, provided such modification or elongation does not eliminate itsability to bind fibrin.

MRI contrast reagents prepared according to the disclosures herein maybe used in the same manner as conventional MRI contrast reagents. Whenimaging a thrombus, certain MR techniques and pulse sequences may bepreferred to enhance the contrast of the thrombus to the backgroundblood and tissues. These techniques include (but are not limited to),for example, black blood angiography sequences that seek to make blooddark, such as fast spin echo sequences (see, e.g., Alexander et al.,Magnetic Resonance in Medicine, 40(2): 298-310 (1998)) and flow-spoiledgradient echo sequences (see, e.g., Edelman et al., Radiology, 177(1):45-50 (1990)). These methods also include flow independent techniquesthat enhance the difference in contrast due to the T₁ difference ofcontrast-enhanced thrombus and blood and tissue, such asinversion-recovery prepared or saturation-recovery prepared sequencesthat will increase the contrast between thrombus and background tissues.In addition, since the present invention does not significantly alterT₂, methods of T₂ preparation may also prove useful (see, e.g., Gronaset al., Journal of Magnetic Resonance Imaging, 7(4): 637-643 (1997)).Finally, magnetization transfer preparations may also improve contrastwith these agents (see, e.g., Goodrich et al., Investigative Radiology,31(6): 323-32 (1996)).

The labeled reagent is administered to the patient in the form of aninjectable composition. The method of administering the MRI contrastagent is preferably parenterally, meaning intravenously,intraarterially, intrathecally, interstitially, or intracavitarilly. Forimaging thrombi, intravenous or intraarterial administration ispreferred. For MRI, it is contemplated that the subject will receive adosage of contrast agent sufficient to enhance the MR signal at the siteof a thrombus at least 10%. After injection with the fibrin bindingmoiety-containing MRI reagent, the patient is scanned in the MRI machineto determine the location of any thrombi. In therapeutic settings, uponthrombus localization, a thrombolytic can be immediately administered,if necessary, and the patient can be subsequently scanned to visualizethrombus degradation.

Ultrasound Imaging

When ultrasound is transmitted through a substance, the acousticproperties of the substance will depend upon the velocity of thetransmissions and the density of the substance. Changes in the acousticproperties will be most prominent at the interface of differentsubstances (solids, liquids, gases). Ultrasound contrast agents areintense sound wave reflectors because of the acoustic differencesbetween liquid (e.g., blood) and gas-containing microvesicles, such asmicrobubbles or microballoons, liposomes, or microspheres dissolvedtherein. Because of their size, ultrasound microvesicles, liposomes,microspheres, and the like may remain for a longer time in the bloodstream after injection than other detectable moieties; a targetedfibrin-specific ultrasound agent therefore may demonstrate superiorimaging of thrombi and sites of angiogenesis.

In this aspect of the invention, the fibrin binding moiety may be linkedto a material which is useful for ultrasound imaging. The materials areemployed to form microvesicles (e.g., liposomes, microbubbles,microspheres, or emulsions) containing a liquid or gas which functionsas the detectable label (e.g., an echogenic gas or material capable ofgenerating an echogenic gas). Materials for the preparation of suchmicrovesicles include amphiphilic compounds such as surfactants, lipids,sphingolipids, oligolipids, or phospholipids, proteins, polypeptides,carbohydrates, and synthetic or natural polymeric materials. See, forfurther description of suitable materials and methods, WO 98/53857, WO98/18498, WO 98/18495, WO 98/18497, WO 98/18496, and WO 98/18501,incorporated herein by reference in their entirety.

For contrast agents comprising suspensions of stabilized microbubbles (apreferred embodiment), amphiphilic components are preferred. Suitableamphiphilic components include phospholipids; lysophospholipids; fattyacids, such as palmitic acid, stearic acid, arachidonic acid or oleicacid; lipids bearing polymers, such as chitin, hyaluronic acid,polyvinylpyrrolidone or polyethylene glycol (PEG), also referred as“pegylated lipids”; lipids bearing sulfonated mono- di-, oligo- orpolysaccharides; cholesterol, cholesterol sulfate or cholesterolhemisuccinate; tocopherol hemisuccinate; lipids with ether orester-linked fatty acids; polymerized lipids; diacetyl phosphate;dicetyl phosphate; ceramides; polyoxyethylene fatty acid esters (such aspolyoxyethylene fatty acid stearates), polyoxyethylene fatty alcohols,polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fattyacid esters, glycerol polyethylene glycol ricinoleate, ethoxylatedsoybean sterols, ethoxylated castor oil or ethylene oxide (EO) andpropylene oxide (PO) block copolymers; sterol aliphatic acid estersincluding, cholesterol butyrate, cholesterol iso-butyrate, cholesterolpalmitate, cholesterol stearate, lanosterol acetate, ergosterolpalmitate, or phytosterol n-butyrate; sterol esters of sugar acidsincluding cholesterol glucuronides, lanosterol glucoronides,7-dehydrocholesterol glucoronide, ergosterol glucoronide, cholesterolgluconate, lanosterol gluconate, or ergosterol gluconate; esters ofsugar acids and alcohols including lauryl glucoronide, stearoylglucoronide, myristoyl glucoronide, lauryl gluconate, myristoylgluconate, or stearoyl gluconate; esters of sugars with aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid or polyuronic acid; saponinsincluding sarsasapogenin, smilagenin, hederagenin, oleanolic acid, ordigitoxigenin; glycerol or glycerol esters including glyceroltripalmitate, glycerol distearate, glycerol tristearate, glyceroldimyristate, glycerol trimyristate, glycerol dilaurate, glyceroltrilaurate, glycerol dipalmitate, long chain alcohols including n-decylalcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, or n-octadecylalcohol; 6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-β-D-mannopyranoside;12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)octadecanoicacid;N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoyl]-2-aminopalmiticacid; N-succinyldioleylphosphatidylethanolamine;1,2-dioleyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinyl-glycerol;1-hexadecyl-2-palmitoylglycerophosphoethanolamine orpalmitoylhomocysteine; alkylamines or alkylammonium salts, comprising atleast one (C₁₀-C₂₀), preferably (C₁₄-C₁₈), alkyl chain, such as, forinstance, N-stearylamine, N,N′-distearylamine, N-hexadecylamine,N,N′-dihexadecylamine, N-stearylammonium chloride,N,N′-distearylammonium chloride, N-hexadecylammonium chloride,N,N′-dihexadecylammonium chloride, dimethyldioctadecylammonium bromide(DDAB), hexadecyltrimethylammonium bromide (CTAB); tertiary orquaternary ammonium salts comprising one or preferably two (C₁₀-C₂₀),preferably (C₁₄-C₁₈), acyl chain linked to the N-atom through a (C₃-C₆)alkylene bridge, such as, for instance,1,2-distearoyl-3-trimethylammonium-propane (DSTAP),1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),1,2-oleoyl-3-trimethylammonium-propane (DOTAP),1,2-distearoyl-3-dimethylammonium-propane (DSDAP); and mixtures orcombinations thereof.

According to a preferred embodiment, at least one of the compoundsforming the microbubbles' envelope is a phospholipid, optionally inadmixture with any of the other above-cited materials. According to thepresent description, the term “phospholipids” is intended to encompassany amphiphilic phospholipid compound, the molecules of which arecapable of forming a stabilizing film of material (typically in the formof a mono-molecular layer) at the gas-water boundary interface in thefinal microbubbles' suspension. Accordingly, these materials are alsoreferred to in the art as “film-forming phospholipids”.

Amphiphilic phospholipid compounds typically contain at least onephosphate group and at least one, preferably two, lipophilic long-chainhydrocarbon groups.

Examples of suitable phospholipids include esters of glycerol with oneor preferably two residues of fatty acids (the same or different) andphosphoric acid, wherein the phosphoric acid residue is in turn bondedto a hydrophilic group, such as, for example, choline(phosphatidylcholines—PC), serine (phosphatidylserines—PS), inositol(phosphatidylinositol), glycerol (phosphatidylglycerols—PG),ethanolamine (phosphatidylethanolamines—PE), and the like groups. Estersof phospholipids with only one residue of fatty acid are generallyreferred to in the art as the “lyso” forms of the phospholipids orlysophospholipids. Fatty acids present in the phospholipids are ingeneral long chain aliphatic acids, typically containing from 12 to 24carbon atoms, preferably from 14 to 22, the aliphatic chain mat containone or more unsaturations or is preferably completely saturated.Examples of suitable fatty acids included in the phospholipids include,for example, lauric acid, myristic acid, palmitic acid, stearic acid,arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenicacid. Preferably, saturated fatty acids such as myristic acid, pamiticacid, stearic acid and arachidic acid are employed.

Further examples of phospholipids are phosphatidic acids, i.e., thediesters of glycerol-phosphoric acid with fatty acids; sphingolipidssuch as sphingomyelins, i.e., those phosphatidylcholine analogs wherethe residue of glycerol diester with fatty acids is replaced by aceramide chain, cardiolipins, i.e. the esters of1,3-diphosphatidylglycerol with a fatty acid, glycolipids such asgangliosides, cerebrosides, etc, glucolipids, sulfatides andglycosphingolipids. As used herein, the term “phospholipids” includeseither naturally occurring, semisynthetic or synthetically preparedproducts that can be employed either singularly or as mixtures. Examplesof naturally occurring phospholipids are natural lecithins(phosphatidylcholine (PC) derivatives) such as, typically, soya bean oregg yolk lecithins.

Examples of semisynthetic phospholipids are the partially or fullyhydrogenated derivatives of the naturally occurring lecithins.

Examples of synthetic phospholipids, which are a preferred embodimentare e.g., dilauryloyl-phosphatidylcholine (“DLPC”),dimyristoylphosphatidylcholine (“DMPC”), dipalmitoyl-phosphatidylcholine(“DPPC”), diarachidoylphosphatidylcholine (“DAPC”),distearoyl-phosphatidylcholine (“DSPC”),1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”),1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”),1-palmitoyl-2-stearoylphosphatidylcholine (“PSPC”),1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”),dioleoylphosphatidylycholine (“DOPC”), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts,diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts,dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts,dipalmitoyl-phosphatidylglycerol (“DPPG”) and its alkali metal salts,distearolyphosphatidylglycerol (“DSPG”) and its alkali metal salts,dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts,dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts,dipalmitoyl phosphatidic acid (“DPPA”) and its alkali metal salts,distearoyl phosphatidic acid (“DSPA”), diarachidoyl phosphatidic acid(“DAPA”) and its alkali metal salts, dimyristoylphosphatidyl-ethanolamin-e (“DMPE”), dipalmitoylphosphatidylethanolamine (“DPPE”), distearoyl phosphatidyl-ethanolamine(“DSPE”), dimyristoyl phosphatidylserine (“DMPS”), diarachidoylphosphatidylserine (“DAPS”), dipalmitoyl phosphatidylserine (“DPPS”),distearoylphosphatidylserine (“DSPS”), dioleoylphosphatidylserine(“DOPS”), dipalmitoyl sphingomyelin (“DPSP”), and distearoylsphingomyelin (“DSSP”).

Preferred phospholipids are fatty acid di-esters of phosphatidylcholine,ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid,phosphatidylethanolamine, phosphatidylserine, phophatidylinositol or ofsphingomyelin. Particularly preferred phospholipids are DAPC, DSPC,DPPA, DSPA, DMPS, DPPS, DSPS and ethyl-DSPC. Most preferred are DPPS orDSPC. Mixtures of phospholipids can also be used, such as, for instance,mixtures of DSPE, DPPE, DPPC, DSPC and/or DAPC with DSPS, DPPS, DSPA,DPPA, DSPG, DPPG, Ethyl-DSPC and/or Ethyl-DPPC.

Suitable phospholipids further include phospholipids modified by linkinga hydrophilic polymer, such as PEG or polypropyleneglycol (PPG) thereto.Phospholipids modified by linking PEG thereto may be referred to hereinas pegylated phospholipids. Examples of modified phospholipids arephosphatidylethanolamines (PE) modified with polyethylenglycol (PEG),“PE-PEGs”, i.e. phosphatidylethanolamines where the hydrophilicethanolamine moiety is linked to a PEG molecule of variable molecularweight (e.g. from 300 to 5000 daltons), such as DPPE-PEG, DSPE-PEG,DMPE-PEG or DAPE-PEG (where DAPE is1,2-diarachidoyl-sn-glycero-3-phosphoethanolamine.) The compositionsalso may contain other amphiphilic compounds including, for instance,fatty acids, such as palmitic acid, stearic acid, arachidonic acid oroleic acid; sterols, such as cholesterol, or esters of sterols withfatty acids or with sugar acids; glycerol or glycerol esters includingglycerol tripalmitate, glycerol distearate, glycerol tristearate,glycerol dimyristate, glycerol trimyristate, glycerol dilaurate,glycerol trilaurate, glycerol dipalmitate; tertiary or quaternaryalkyl-ammonium salts, such as 1,2-distearoyl-3-trimethylammonium-propane(DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), andmixtures or combinations thereof.

Preferably, the formulation (and particularly the microbubble envelope)includes at least on component bearing an overall net charge, such as,for instance, a charged amphiphilic material, preferably a lipid or aphospholipid. Examples of phospholipids bearing an overall negativecharge are derivatives, in particular fatty acid di-ester derivatives,of phosphatidylserine, such as DMPS, DPPS, DSPS; of phosphatidic acid,such as DMPA, DPPA, DSPA; of phosphatidylglycerol such as DMPG, DPPG andDSPG or of phosphatidylinositol, such as DMPI, DPPI or DPPI. Alsomodified phospholipids, in particular PEG-modifiedphosphatidylethanolamines, such as DPPE-PEG or DSPE-PEG, can be used asnegatively charged molecules. Also the lyso-form of the above citedphospholipids, such as lysophosphatidylserine derivatives (e.g.lyso-DMPS, -DPPS or -DSPS), lysophosphatidic acid derivatives (e.g.lyso-DMPA, -DPPA or -DSPA) and lysophosphatidylglycerol derivatives(e.g. lyso-DMPG, -DPPG or -DSPG), can advantageously be used asnegatively charged compounds. Other examples of negatively chargedcompounds are bile acid salts such as cholic acid salts, deoxycholicacid salts or glycocholic acid salts; and (C₁₂-C₂₄), preferably(C₁₄-C₂₂) fatty acid salts such as, for instance, palmitic acid salts,stearic acid salts, 1,2-dipalmitoyl-sn-3-succinylglycerol salts or1,3-dipalmitoyl-2-succinylglycerol salts.

Preferably, the negatively charged compound is selected among DPPA,DPPS, DSPG, DSPE-PEG2000, DSPE-PEG5000 or mixtures thereof.

The negatively charged component is typically associated with acorresponding positive counter-ion, which can be mono- (e.g. an alkalimetal or ammonium), di- (e.g. an alkaline earth metal) or tri-valent(e.g. aluminium). Preferably the counter-ion is selected among alkalimetal cations, such as Li⁺, Na⁺, or K⁺, more preferably Na⁺.

Examples of phospholipids bearing an overall positive charge arederivatives of ethylphosphatidylcholine, in particular di-esters ofethylphosphatidylcholine with fatty acids, such as1,2-distearoyl-sn-glycero-3-ethylphosphocholine (Ethyl-DSPC or DSEPC),1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (Ethyl-DPPC or DPEPC).The negative counterion is preferably a halide ion, in particularchloride or bromide ion. Examples of positively charged compounds thatcan be incorporated into the envelope of microbubbles are mono-, di-tri-, or tetra-alkylammonium salts with a halide counter ion (e.g.chloride or bromide) comprising at least one (C₁₀-C₂₀), preferably(C₁₄-C₁₈), alkyl chain, such as, for instance mono- ordi-stearylammonium chloride, mono or di-hexadecylammonium chloride,dimethyldioctadecylammonium bromide (DDAB) or hexadecyltrimethylammoniumbromide (CTAB). Further examples of positively charged compounds thatcan be incorporated into the envelope of microbubbles are tertiary orquaternary ammonium salts with a halide counter ion (e.g. chloride orbromide) comprising one or preferably two (C₁₀-C₂₀), preferably(C₁₄-C₁₈), acyl chains linked to the N-atom through a (C₃-C₆) alkylenebridge, such as, for instance,1,2-distearoyl-3-trimethylammonium-propane (DSTAP),1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),1,2-oleoyl-3-trimethylammonium-propane (DOTAP) or1,2-distearoyl-3-dimethylammonium-propane (DSDAP).

DSEPC, DPEPC and/or DSTAP are preferably employed as positively chargedcompounds in the microbubble envelope.

The positively charged component is typically associated with acorresponding negative counter-ion, which can be mono- (e.g. halide),di- (e.g. sulphate) or tri-valent (e.g. phosphate). Preferably thecounter-ion is selected from among the halide ions, such as F⁻(fluorine), Cl⁻ (chlorine) or Br⁻ (bromine).

Mixtures of neutral and charged compounds, in particular ofphospholipids and/or lipids, can be satisfactorily employed to form themicrobubble envelope. The amount of charged lipid or phospholipid mayvary from about 95 mol % to about 1 mol %, with respect to the totalamount of lipid and phospholipid, preferably from 80 mol % to 20 mol %.

Preferred mixtures of neutral phospholipids and charged lipids orphospholipids are, for instance, DPPG/DSPC, DSTAP/DAPC, DPPS/DSPC,DPPS/DAPC, DPPE/DPPG, DSPA/DAPC, DSPA/DSPC and DSPG/DSPC.

In preferred embodiments, the phospholipid is the main component of thestabilizing envelope of the microbubbles, amounting to at least 50%(w/w) of the total amount of components forming the envelope of thegas-filled microbubbles. In some preferred embodiments, substantiallythe totality of the envelope (at least 90% and up to 100% by weight) canbe formed of the phospholipid.

Any of the gases disclosed herein or known to the skilled artisan may beemployed; however, inert gases, such as SF₆ or perfluorocarbons likeCF₄, C₃F₈ and C₄F₁₀, are preferred, optionally in admixture with othergases such as air, nitrogen, oxygen or carbon dioxide

The preferred microvesicle suspensions of the present invention may beprepared from phospholipids using known processes, such as, for example,freeze-drying or spray-drying solutions of the crude phospholipids in asuitable solvent or using the processes set forth in EP 554213; U.S.Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962; EP 682530; U.S.Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; U.S. Pat. No. 6,183,725; EP474833; U.S. Pat. No. 5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No.5,531,980; U.S. Pat. No. 5,567,414; U.S. Pat. No. 5,658,551; U.S. Pat.No. 5,643,553; U.S. Pat. No. 5,911,972; U.S. Pat. No. 6,110,443; U.S.Pat. No. 6,136,293; EP 619743; U.S. Pat. No. 5,445,813; U.S. Pat. No.5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat. No. 6,187,288; and U.S.Pat. No. 5,908,610, which are incorporated by reference herein in theirentirety. Preferably, the phospholipids are dissolved in an organicsolvent and the solution is dried without going through a liposomeformation stage. This can be done by dissolving the phospholipids in asuitable organic solvent together with a hydrophilic stabilizersubstance or a compound soluble both in the organic solvent and waterand freeze-drying or spray-drying the solution. In this embodiment thecriteria used for selection of the hydrophilic stabilizer is itssolubility in the organic solvent of choice. Examples of hydrophilicstabilizer compounds soluble in water and the organic solvent are, e.g.,a polymer, like polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA),polyethylene glycol (PEG), etc., malic acid, glycolic acid, maltol, andthe like. Such hydrophilic compounds also aid in homogenizing themicrobubbles size distribution and enhance stability under storage. Anysuitable organic solvent may be used as long as its boiling point issufficiently low and its melting point is sufficiently high tofacilitate subsequent drying. Typical organic solvents include, forexample, dioxane, cyclohexanol, tertiary butanol, tetrachlorodifluoroethylene (C₂Cl₄F₂) or 2-methyl-2-butanol. 2-methyl-2-butanol and C₂Cl₄F₂are preferred.

Prior to formation of the suspension of microvesicles by dispersion inan aqueous carrier, the freeze dried or spray dried phospholipid powdersare contacted with air or another gas. When contacted with the aqueouscarrier the powdered phospholipids whose structure has been disruptedwill form lamellarized or laminarized segments that will stabilize themicrobubbles of the gas dispersed therein. This method permitsproduction of suspensions of microbubbles that are stable even whenstored for prolonged periods and are obtained by simple dissolution ofthe dried laminarized phospholipids (which have been stored under adesired gas) without shaking or any violent agitation.

Alternatively, microvesicles, and particularly microbubbles, can beprepared by suspending a gas into an aqueous solution at high agitationspeed, as disclosed e.g. in WO 97/29783. Preferably, as disclosed inInternational patent application WO 04/069284, a microemulsion can beprepared which contains the phospholipids (e.g., DSPC and/or DSPA) inadmixture with a lyoprotecting agent (such as, for instance,carbohydrates, sugar alcohols, polyglycols and mixtures thereof, asindicated in detail hereinafter) and optionally other amphiphilicmaterials (such as stearic acid), dispersed in an emulsion of water andof a water immiscible organic solvent. Preferred organic solvents arethose having solubility in water of 1.0 g/1 or lower, preferably lowerabout 0.01 g/l, and include, for instance, pentane, hexane, heptane,octane, nonane, decane, 1-pentene, 2-pentene, 1-octene, cyclopentane,cyclohexane, cyclooctane, 1-methyl-cyclohexane, benzene, toluene,ethylbenzene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, di-butyl etherand di-isopropylketone, chloroform, carbon tetrachloride,2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane),2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane),tetrachloro-1,1-difluoroethane, perfluoropentane, perfluorohexane,perfluoroheptane, perfluorononane, perfluorobenzene, perfluorodecalin,methylperfluorobutylether, methylperfluoroisobutylether,ethylperfluorobutylether, ethylperfluoroisobutylether and mixturesthereof. The fibrin binding peptide of the invention conjugated to aphospholipid (e.g. the lipopeptides discussed herein) can be admixedtogether with the phospholipid forming the microvesicle's envelope, inthe microemulsion. Preferably, an aqueous suspension of the fibrinbinding peptide-phospholipid conjugate and of a PE-PEG (e.g.DSPE-PEG2000) is first prepared, which is then admixed together with anaqueous-organic emulsion comprising the phospholipid and thelyoprotecting agent. Preferably said mixing is effected under heating,e.g. form about 40° C. to 80° C.

Other excipients or additives may be present either in the dryformulation of the microbubbles or may be added together with theaqueous carrier used for the reconstitution thereof, without necessarilybeing involved (or only partially involved) in the formation of thestabilizing envelope of the microbubble. These include pH regulators,osmolality adjusters, viscosity enhancers, emulsifiers, bulking agents,etc. and may be used in conventional amounts. For instance compoundslike polyoxypropylene glycol and polyoxyethylene glycol as well ascopolymers thereof can be used. Examples of viscosity enhancers orstabilizers are compounds selected from linear and cross-linked poly-and oligo-saccharides, sugars and hydrophilic polymers such aspolyethylene glycol.

As the preparation of gas-filled microbubbles may involve a freezedrying or spray drying step, it may be advantageous to include in theformulation a lyophilization additive, such as an agent withcryoprotective and/or lyoprotective effect and/or a bulking agent, forexample an amino-acid such as glycine; a carbohydrate, e.g. a sugar suchas sucrose, mannitol, maltose, trehalose, glucose, lactose or acyclodextrin, or a polysaccharide such as dextran; or apolyoxyalkyleneglycol such as polyethylene glycol.

In ultrasound applications the contrast agents formed by phospholipidstabilized microbubbles can be administered, for example, in doses suchthat the amount of phospholipid injected is in the range 0.1 to 200μg/kg body weight, preferably from about 0.1 to 30 μg/kg.

Other gas containing suspensions include those disclosed in, forexample, U.S. Pat. No. 5,798,091, WO 97/29783, also EP 881 915,incorporated herein by reference in their entirety. These agents may beprepared as described in U.S. Pat. No. 5,798,091 or WO97/29783.

Another preferred ultrasound contrast agent comprises ultrasoundcontrast agents in the form of “microballoons” of “microcapsules”. Theterms “microballoon” or “microcapsules” (here used interchangeably)refer to gas filled bodies with a material boundary or envelope. More onmicroballoon formulations and methods of preparation may be found in EP324 938 (U.S. Pat. No. 4,844,882); U.S. Pat. No. 5,711,933; U.S. Pat.No. 5,840,275; U.S. Pat. No. 5,863,520; U.S. Pat. No. 6,123,922; U.S.Pat. No. 6,200,548; U.S. Pat. No. 4,900,540; U.S. Pat. No. 5,123,414;U.S. Pat. No. 5,230,882; U.S. Pat. No. 5,469,854; U.S. Pat. No.5,585,112; U.S. Pat. No. 4,718,433; U.S. Pat. No. 4,774,958; WO95/01187; U.S. Pat. No. 5,529,766; U.S. Pat. No. 5,536,490; and U.S.Pat. No. 5,990,263, the contents of which are incorporated herein byreference.

The preferred microballoons have an envelope including a biodegradablephysiologically compatible polymer or, a biodegradable solid lipid. Thepolymers useful for the preparation of the microballoons of the presentinvention can be selected from the biodegradable physiologicallycompatible polymers, such as any of those described in any of thefollowing patents: EP 458745, U.S. Pat. No. 5,711,933, U.S. Pat. No.5,840,275, EP 554213, U.S. Pat. No. 5,413,774 and U.S. Pat. No.5,578,292, the entire contents of which are incorporated herein byreference. In particular, the polymer can be selected from biodegradablephysiologically compatible polymers, such as polysaccharides of lowwater solubility, polylactides and polyglycolides and their copolymers,copolymers of lactides and lactones such as .epsilon.-caprolactone,.gamma.-valerolactone and polypeptides. Other suitable polymers includepoly(ortho)esters (see e.g., U.S. Pat. No. 4,093,709; U.S. Pat. No.4,131,648; U.S. Pat. No. 4,138,344; U.S. Pat. No. 4,180,646); polylacticand polyglycolic acid and their copolymers, for instance DEXON (see J.Heller, Biomaterials 1 (1980), 51;poly(DL-lactide-co-.epsilon.-caprolact-one),poly(DL-lactide-co-.gamma.-valerolactone),poly(DL-lactide-co-.gamma-.-butyrolactone), polyalkylcyanoacrylates;polyamides, polyhydroxybutyrate; polydioxanone; poly-.beta.-aminoketones(A. S. Angeloni, P. Ferruti, M. Tramontini and M. Casolaro. The Mannichbases in polymer synthesis: 3. Reduction of poly(beta-aminoketone(s)) topoly(gamma-aminoalcohol(s)) and their N-alkylation topoly(gamma-hydroxyquaternary ammonium salt(s)), Polymer 23, pp1693-1697, 1982.); polyphosphazenes (Allcock, Harry R. Polyphosphazenes:new polymers with inorganic backbone atoms (Science 193:1214-19 (1976))and polyanhydrides. The microballoons of the present invention can alsobe prepared according to the methods of WO-A-96/15815, incorporatedherein by reference, where the microballoons are made from abiodegradable membrane comprising biodegradable lipids, preferablyselected from mono- di-, tri-glycerides, fatty acids, sterols, waxes andmixtures thereof. Preferred lipids are di- or tri-glycerides, e.g., di-or tri-myristin, -palmityn or -stearin, in particular tripalmitin ortristearin. The microballoons may employ any of the gases disclosedherein of known to the skilled artisan; however, inert gases such asfluorinated gases are preferred. The microballoons may be suspended in apharmaceutically acceptable liquid carrier with optional additives knownto those of ordinary skill in the art and stabilizers.

Other gas-containing contrast agent formulations include microparticles(especially aggregates of microparticles) having gas contained thereinor otherwise associated therewith (for example being adsorbed on thesurface thereof and/or contained within voids, cavities or porestherein). Methods for the preparation of these agents are as describedin EP 0122624; EP 0123235; EP 0365467; U.S. Pat. No. 5,558,857; U.S.Pat. No. 5,607,661; U.S. Pat. No. 5,637,289; U.S. Pat. No. 5,558,856;U.S. Pat. No. 5,137,928; WO 95/21631 or WO 93/13809, incorporated hereinby reference in their entirety.

Any of these ultrasound compositions should also be, as far as possible,isotonic with blood. Hence, before injection, small amounts of isotonicagents may be added to any of above ultrasound contrast agentsuspensions. The isotonic agents are physiological solutions commonlyused in medicine and they comprise aqueous saline solution (0.9% NaCl),2.6% glycerol solution, 5% dextrose solution, etc. Additionally, theultrasound compositions may include standard pharmaceutically acceptableadditives, including, for example, emulsifying agents, viscositymodifiers, cryoprotectants, lyoprotectants, bulking agents etc.

Any biocompatible gas may be used in the ultrasound contrast agents ofthe invention. The term “gas” as used herein includes any substances(including mixtures) substantially in gaseous form at the normal humanbody temperature. The gas may thus include, for example, air, nitrogen,oxygen, CO₂, hydrogen, nitrous oxide, a noble or inert gas such ashelium, argon, xenon or krypton, a radioactive gas such as Xe¹³³ orKr⁸¹, a hyperpolarized noble gas such as hyperpolarized helium, xenon orneon, fluorinated gases (including for example, perfluorocarbons, SF₆,SeF₆) a low molecular weight hydrocarbon (e.g., containing from 1 to 7carbon atoms), for example, an alkane such as methane, ethane, apropane, a butane, isobutene, isopentane or pentane, a cycloalkane suchas cyclobutane or cyclopentane, an alkene such as propene, propadiene ora butene, or an alkyne such as acetylene, an ether, a ketone, an ester,halogenated gases, such as halogenated, fluorinated or perfluorinatedlow molecular weight hydrocarbons (e.g. containing up to 7 carbon atoms)and/or mixtures thereof.

Fluorinated gases are preferred, in particular perfluorinated gases.Fluorinated gases include materials that contain at least one fluorineatom such as SF₆, freons (organic compounds containing one or morecarbon atoms and fluorine, i.e., CF₄, C₂F₆, C₃F₈, C₄F₈, C₄F₁₀, CBrF₃,CCl₂F₂, C₂ClF₅, and CBrClF₂), fluorinated hydrocarbons, fluorinatedketones such as perfluoroacetone, fluorinated ethers such asperfluorodiethyl ether and perfluorocarbons. The term perfluorocarbonrefers to compounds containing only carbon and fluorine atoms andincludes, in particular, saturated, unsaturated, and cyclicperfluorocarbons. The saturated perfluorocarbons, which are usuallypreferred, have the formula C_(n)F_(n)+2, where n is from 1 to 12,preferably from 2 to 10, most preferably from 3 to 8 and even morepreferably from 3 to 6. Examples of biocompatible, physiologicallyacceptable perfluorocarbons are: perfluoroalkanes, such asperfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes(e.g. perfluoro-n-butane, optionally in admixture with other isomerssuch as perfluoro-isobutane), perfluoropentanes, perfluorohexanes orperfluoroheptanes; perfluoroalkenes, such as perfluoropropene,perfluorobutenes (e.g. perfluorobut-2ene) or perfluorobutadiene;perfluoroalkynes (e.g. perfluorobut-2-yne); and perfluorocycloalkanes(e.g. perfluorocyclobutane, perfluoromethylcyclobutane,perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes,perfluorocyclopentane, perfluoromethylcyclopentane,perfluorodimethylcyclopentanes, perfluorocyclohexane,perfluoromethylcyclohexane and perfluorocycloheptane). Preferredsaturated perfluorocarbons include, for example, CF₄, C₂F₆, C₃F₈, C₄F₈,C₄F₁₀, C₅F₁₂ and C₆F₁₂. Most preferably the gas or gas mixture comprisesSF₆ or a perfluorocarbon selected from the group consisting of C₃F₈C₄F₈, C₄F₁₀, C₅F₂, C₆F₁₂, C₇F₁₄, C₈F₁₈, with C₄F₁₀ being particularlypreferred. See also WO 97/29783, WO 98/53857, WO 98/18498, WO 98/18495,WO 98/18496, WO 98/18497, WO 98/18501, WO 98/05364, WO 98/17324.

In certain circumstances it may be desirable to include a precursor to agaseous substance (e.g., a material that is capable of being convertedto a gas in vivo, often referred to as a “gas precursor”). Preferablythe gas precursor and the gas it produces are physiologicallyacceptable. The gas precursor may be pH-activated, photo-activated,temperature activated, etc. For example, certain perfluorocarbons may beused as temperature activated gas precursors. These perfluorocarbons,such as perfluoropentane, have a liquid/gas phase transition temperatureabove room temperature (or the temperature at which the agents areproduced and/or stored) but below body temperature; thus they undergo aphase shift and are converted to a gas within the human body.

As discussed above, the gas can comprise a mixture of gases. The mixturemay comprise any of the above gases in any ratio. In one preferredembodiment, the mixture may include a conventional gas such as nitrogen,air or carbon dioxide and a fluorinated as. Examples of suitable gasmixtures can be found in WO 94/09829, which is herein incorporated byreference. The following combinations are particularly preferred gasmixtures: a mixture of gases (A) and (B) in which, at least one of thegases (B), present in an amount of between 0.5-41% by vol., has amolecular weight greater than 80 daltons and is a fluorinated gas and(A) is selected from the group consisting of air, oxygen, nitrogen,carbon dioxide and mixtures thereof, the balance of the mixture beinggas A.

For the use in MRI the microvesicles will preferably contain ahyperpolarized noble gas such as hyperpolarized neon, hyperpolarizedhelium, hyperpolarized xenon, or mixtures thereof, optionally inadmixture with air, CO₂, oxygen, nitrogen, helium, xenon, or any of thehalogenated hydrocarbons as defined above.

For use in scintigraphy, the microvesicle will preferably containradioactive gases such as Xe¹³³ or Kr⁸¹ or mixtures thereof, optionallyin admixture with air, CO₂, oxygen, nitrogen, helium, krypton or any ofthe halogenated hydrocarbons as defined above.

Since ultrasound vesicles may be larger than the other detectable labelsdescribed herein, in one preferred embodiment they are be linked orconjugated to a plurality of fibrin-binding polypeptides in order toincrease the targeting efficiency of the agent. Attachment to theultrasound contrast agents described above (or known to those skilled inthe art) may be via direct covalent bond between the fibrin-bindingpolypeptide and the material used to make the vesicle or via a linker,as described previously. For example, see WO 98/53857 generally for adescription of the attachment of a peptide to a bifunctional PEG linker,which is then reacted with a liposome composition. See also, Lanza etal., Ultrasound in Med. & Bio., 23(6):863-870 (1997).

A number of methods may be used to prepare suspensions of microvesiclesconjugated to fibrin-binding polypeptides. For example, one may preparemaleimide-derivatized microbubbles by incorporating 5% (w/w) of N-MPB—PE(1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine-4-(p-maleimido-phenylbutyramide), (Avanti Polar-Lipids, Inc) in the phospholipid formulation.Then, solutions of mercaptoacetylated fibrin-binding peptides (10 mg/mLin DMF), which have been incubated in deacetylation solution (50 mMsodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCl, pH 7.5) are addedto the maleimide-activated microbubble suspension. After incubation inthe dark, under gentle agitation, the peptide conjugated microbubblesmay be purified by centrifugation.

Compounds that can be used for derivatization of microvesicles andparticularly microbubbles typically include the following components:(a) a hydrophobic portion, compatible with the material forming theenvelope of the microbubble or of the microballoon, in order to allow aneffective incorporation of the compound in the envelope of the vesicle;said portion is represented typically by a lipid moiety (dipalmitin,distearoyl); and (b) a spacer (typically PEGs of different molecularweights, an amino acid chain, etc.), which may be optional in some cases(for example, microbubbles may for instance present difficulties to befreeze dried if the spacer is too long) or preferred in some others(e.g., peptides may be less active when conjugated to a microballoonwith short spacers); and (c) a reactive group capable of reacting with acorresponding reacting moiety on the peptide to be conjugated (e.g.,maleimido with the —SH group of cysteine).

Alternatively, fibrin-binding polypeptide conjugated microbubbles may beprepared using biotin/avidin. For example, avidin-conjugatedmicrobubbles may be prepared using a maleimide-activated phospholipidmicrobubble suspension, prepared as described above, which is added tomercaptoacetylated-avidin (which has been incubated with deacetylationsolution). Biotinylated fibrin-binding peptides are then added to thesuspension of avidin-conjugated microbubbles, yielding a suspension ofmicrobubbles conjugated to fibrin-binding peptides.

Additionally fibrin binding peptides may be conjugated to phospholipids,these lipopeptides may then be used to prepare gas filled microvesicleultrasound contrast agents. Preferably, the phospholipid may be selectedfrom the group consisting of: phosphatidylethanolamines and modifiedphosphatidylethanolamines. The peptide and the phospholipid may beconjugated directly or via a linker, including for example a hydrophilicpolymer, an amino acid chain, etc. Particularly preferred phospholipidsinclude phosphatidylethanolamines modified by linking a hydrophilicpolymer thereto. Examples of modified phosphatidylethanolamines arephosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), inbrief “PE-PEGs”, i.e. phosphatidylethanolamines where the hydrophilicethanolamine moiety is linked to a PEG molecule of variable molecularweight (e.g. from 300 to 5000 daltons), such as DPPE-PEG, DSPE-PEG,DMPE-PEG or DAPE-PEG. DSPE-PEG2000, DSPE-PEG3400, DPPE-PEG2000 andDPPE-PEG3400 are preferred, with DSPE-PEG2000 particularly preferred.Note that a salt form of the phospholipid may be used, such as, forexample, the trimethyl ammonium salt, the tetramethylammonium salt, thetriethylammonium salt, sodium salt, etc. Methods of preparing suchlipopeptides are set forth in the examples.

Some preferred methods of preparing targeted microvesicles withfibrin-binding polypeptides conjugated to phospholipids are included inthe examples.

Unless it contains a hyperpolarized gas, known to require specialstorage conditions, the lyophilized residue may be stored andtransported without need of temperature control of its environment andin particular it may be supplied to hospitals and physicians for on siteformulation into a ready-to-use administrable suspension withoutrequiring such users to have special storage facilities. Preferably insuch a case it can be supplied in the form of a two-component kit, whichcan include two separate containers or a dual-chamber container. In theformer case preferably the container is a conventional septum-sealedvial, wherein the vial containing the lyophilized residue of step b) issealed with a septum through which the carrier liquid may be injectedusing an optionally prefilled syringe. In such a case the syringe usedas the container of the second component is also used then for injectingthe contrast agent. In the latter case, preferably the dual-chambercontainer is a dual-chamber syringe and once the lyophilizate has beenreconstituted and then suitably mixed or gently shaken, the containercan be used directly for injecting the contrast agent. In both casesmeans for directing or permitting application of sufficient bubbleforming energy into the contents of the container are provided. However,as noted above, in the stabilized contrast agents according to theinvention the size of the gas microbubbles is substantially independentof the amount of agitation energy applied to the reconstituted driedproduct. Accordingly, no more than gentle hand shaking is generallyrequired to give reproducible products with consistent microbubble size.

It can be appreciated by one of ordinary skilled in the art that othertwo-chamber reconstitution systems capable of combining the dried powderwith the aqueous solution in a sterile manner are also within the scopeof the present invention. In such systems, it is particularlyadvantageous if the aqueous phase can be interposed between thewater-insoluble gas and the environment, to increase shelf life of theproduct. Where a material necessary for forming the contrast agent isnot already present in the container (e.g. a targeting ligand to belinked to the phospholipid during reconstitution), it can be packagedwith the other components of the kit, preferably in a form or containeradapted to facilitate ready combination with the other components of thekit.

No specific container, vial or connection system is required; thepresent invention may use conventional containers, vials and adapters.The only requirement is a good seal between the stopper and thecontainer. The quality of the seal, therefore, becomes a matter ofprimary concern; any degradation of seal integrity could allowundesirable substances to enter the vial. In addition to assuringsterility, vacuum retention is essential for products stoppered atambient or reduced pressures to assure safe and proper reconstitution.The stopper may be a compound or multicomponent formulation based on anelastomer, such as poly(isobutylene) or butyl rubber.

Ultrasound imaging techniques that can be used in accordance with thepresent invention include known techniques, such as color Doppler, powerDoppler, Doppler amplitude, stimulated acoustic imaging, and two- orthree-dimensional imaging techniques. Imaging may be done in harmonic(resonant frequency) or fundamental modes, with the second harmonicpreferred.

In ultrasound applications the contrast agents formed by phospholipidstabilized microbubbles may, for example, be administered in doses suchthat the amount of phospholipid injected is in the range 0.1 to 200μg/kg body weight, preferably from about 0.1 to 30 μg/kg.Microballoons-containing contrast agents are typically administered indoses such that the amount of wall-forming polymer or lipid is fromabout 10 μg/kg to about 20 mg/kg of body weight.

In a preferred embodiment, the ultrasound contrast agents describedherein are conjugated to one or more fibrin-binding moieties. As shownin the examples, these targeted ultrasound contrast agents will localizeat blood clots containing fibrin, fibrin-containing tissue or sites ofangiogenesis and may be used to image clots, cancer or angiogenictissue.

The ultrasound contrast agents of the present invention may further beused in a variety of therapeutic imaging methods. The term therapeuticimaging includes within its meaning any method for the treatment of adisease in a patient which comprises the use of a contrast imaging agent(e.g. for the delivery of a therapeutic agent to a selected receptor ortissue), and which is capable of exerting or is responsible to exert abiological effect in vitro and/or in vivo. Therapeutic imaging mayadvantageously be associated with the controlled localized destructionof the gas-filled microvesicles, e.g. by means of an ultrasound burst athigh acoustic pressure (typically higher than the one generally employedin non-destructive diagnostic imaging methods). This controlleddestruction may be used, for instance, for the treatment of blood clots(a technique also known as sonothrombolysis), optionally in combinationwith the localized release of a suitable therapeutic agent.Alternatively, said therapeutic imaging may include the delivery of atherapeutic agent into cells, as a result of a transient membranepermeabilization at the cellular level induced by the localized burst ofthe microvesicles. This technique can be used, for instance, for aneffective delivery of genetic material into the cells; optionally, adrug can be locally delivered in combination with genetic material, thusallowing a combined pharmaceutical/genetic therapy of the patient (e.g.in case of tumor treatment).

The term “therapeutic agent” includes within its meaning any substance,composition or particle which may be used in any therapeuticapplication, such as in methods for the treatment of a disease in apatient, as well as any substance which is capable of exerting orresponsible to exert a biological effect in vitro and/or in vivo.Therapeutic agents thus include any compound or material capable ofbeing used in the treatment (including diagnosis, prevention,alleviation, pain relief or cure) of any pathological status in apatient (including malady, affliction, disease lesion or injury).Examples of therapeutic agents include those discussed herein, such as,for example, drugs, pharmaceuticals, bioactive agents, cytotoxic agents,chemotherapy agents, radiotherapeutic agents, proteins, natural orsynthetic peptides, including oligopeptides and polypeptides, vitamins,steroids and genetic material, including nucleosides, nucleotides,oligonucleotides, polynucleotides and plasmids. In a preferredembodiment the therapeutic agent is a drug useful in the treatment ofcancer, thrombotic disorders or angiogenic disorders.

Optical Imaging, Sonoluminescence or Photoacoustic Imaging

In another embodiment, the fibrin binding moieties of the invention maybe conjugated (directly or via a linker) to an optical, sonolumiscent orphotoacoustic label. In a preferred embodiment, the fibrin bindingmoieties of the invention are conjugated (directly or via a linker) toan optically active imaging moiety. Suitable examples of opticallyactive imaging moieties include, for example, optical dyes, includingorganic chromophores or fluorophores, having extensive delocalized ringsystems and having absorption or emission maxima in the range of400-1500 nm; a fluorescent molecule such as, for example, fluorescein; aphosphorescent molecule; a molecule absorbing in the UV spectrum; aquantum dot; or a molecule capable of absorption of near or far infraredradiations. One preferred optically active moiety is5-carboxyfluorescein (CF5).

In accordance with the present invention, a number of optical parametersmay be employed to determine the location of fibrin with in vivo lightimaging after injection of the subject with an optically-labeled fibrinbinding moiety. Optical parameters to be detected in the preparation ofan image may include transmitted radiation, absorption, fluorescent orphosphorescent emission, light reflection, changes in absorbanceamplitude or maxima, and elastically scattered radiation. For example,biological tissue is relatively translucent to light in the nearinfrared (NIR) wavelength range of 650-1000 nm. NIR radiation canpenetrate tissue up to several centimeters, permitting the use of thefibrin binding moieties of the present invention for optical imaging offibrin in vivo.

Near infrared dye may include, cyanine or indocyanine derivatives suchas, for example, Cy5.5, IRDye800, indocyanine green (ICG), indocyaninegreen derivatives including the tetrasulfonic acid substitutedindocyanine green (TS-ICG), and combinations thereof.

The fibrin binding moieties may be conjugated with photolabels, such asoptical dyes, including organic chromophores or fluorophores, havingextensive delocalized ring systems and having absorption or emissionmaxima in the range of 400-1500 nm. The fibrin binding moiety mayalternatively be derivatized with a bioluminescent molecule. Thepreferred range of absorption or emission maxima for photolabels isbetween 600 and 1000 nm to minimize interference with the signal fromhemoglobin. Preferably, photoabsorption labels have large molarabsorptivities, e.g. >10⁵ cm⁻¹M⁻¹, while fluorescent optical dyes willhave high quantum yields. Examples of optical dyes include, but are notlimited to those described in WO 98/18497, WO 98/18496, WO 98/18495, WO98/18498, WO 98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO98/47538, and references cited therein. The photolabels may becovalently linked directly to the fibrin binding moiety or linked to thefibrin binding moiety via a linker, as described previously.

After injection of the optically-labeled fibrin binding moiety, thepatient is scanned with one or more light sources (e.g., a laser) in thewavelength range appropriate for the photolabel employed in the agent.The light used may be monochromatic or polychromatic and continuous orpulsed. Transmitted, scattered, or reflected light is detected via aphotodetector tuned to one or multiple wavelengths to determine thelocation of fibrin in the subject. Changes in the optical parameter maybe monitored over time to detect accumulation of the optically-labeledreagent at the site of the thrombus. Standard image processing anddetecting devices may be used in conjunction with the optical imagingreagents of the present invention.

The optical imaging reagents described above may also be used foracousto-optical or sonoluminescent imaging performed withoptically-labeled imaging agents (see, U.S. Pat. No. 5,171,298, WO98/57666, and references therein). In acousto-optical imaging,ultrasound radiation is applied to the subject and affects the opticalparameters of the transmitted, emitted, or reflected light. Insonoluminescent imaging, the applied ultrasound actually generates thelight detected. Suitable imaging methods using such techniques aredescribed in WO 98/57666.

Additionally, the fibrin-binding moieties of the invention may beattached to an enzyme substrate that is linked to both a light imagingreporter and a light imaging quencher. The fibrin binding moiety servesto localize the construct to the fibrin-bearing tissue of interest (e.ga tumor), where an enzyme cleaves the enzyme substrate, releasing thelight imaging quencher and allowing light imaging of the fibrin-bearingtissue of interest.

Nuclear Imaging (Radionuclide Imaging) and Radiotherapy.

Fibrin-binding moieties also may be conjugated with a radionuclidereporter appropriate for scintigraphy, SPECT, or PET imaging and/or witha radionuclide appropriate for radiotherapy. Constructs in which thefibrin binding moieties are conjugated with both a chelator for aradionuclide useful for diagnostic imaging and a chelator useful forradiotherapy are within the scope of the invention.

For use as a PET agent a peptide is complexed with one of the variouspositron emitting metal ions, such as ⁵¹Mn, ⁵²Fe, ⁶⁰Cu, ⁶⁸Ga, ⁷²As,^(94m)Tc, or ¹¹⁰In. The binding moieties of the invention can also belabeled by halogenation using radionuclides such as ¹⁸F, ¹²⁴I, ¹²⁵I,¹³¹I, ¹²³I, ⁷⁷Br, and ⁷⁶Br. Preferred metal radionuclides forscintigraphy or radiotherapy include ^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc,⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho,¹⁶⁵Dy, ¹⁶⁶Dy, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb,²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb,¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au. The choice of metal will be determined based onthe desired therapeutic or diagnostic application. For example, fordiagnostic purposes the preferred radionuclides include ⁶⁴Cu, ⁶⁷Ga,⁶⁸Ga, ^(99m)Tc, and ¹¹¹In. For therapeutic purposes, the preferredradionuclides include ⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹In, ¹¹⁷mSn, ¹⁴⁹Pm, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁸⁶⁻¹⁸⁸Re, and ¹⁹⁹Au. ^(99m)Tc isparticularly preferred for diagnostic applications because of its lowcost, availability, imaging properties, and high specific activity. Thenuclear and radioactive properties of Tc-99m make this isotope an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a.⁹⁹Mo-⁹⁹mTc generator.

The metal radionuclides may be chelated by a chelators. Suitablechelators include those discussed above, as well as, for example,linear, macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelators,including for example, the ligands disclosed in U.S. Pat. No. 5,367,080,U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556, U.S. Pat. No.5,075,099, and U.S. Pat. No. 5,886,142, and other chelators known in theart including, but not limited to, HYNIC, and bisamino bisthiol (BAT)chelators (see also U.S. Pat. No. 5,720,934). For example, N₄ chelatorsare described in U.S. Pat. No. 6,143,274; U.S. Pat. No. 6,093,382; U.S.Pat. No. 5,608,110; U.S. Pat. No. 5,665,329; U.S. Pat. No. 5,656,254;and U.S. Pat. No. 5,688,487. Certain N₃S chelators are described inPCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. No.5,662,885; U.S. Pat. No. 5,976,495; and U.S. Pat. No. 5,780,006. Thechelator may also include derivatives of the chelating ligandmercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N₃S, andN₂S₂ systems such as MAMA (monoamidemonoaminedith-iols), DADS (N₂Sdiaminedithiols), CODADS and the like. These ligand systems and avariety of others are described in Liu and Edwards, Chem Rev.,99:2235-2268 (1999) and references therein.

The chelator may also include complexes containing ligand atoms that arenot donated to the metal in a tetradentate array. These include theboronic acid adducts of technetium and rhenium dioximes, such as aredescribed in U.S. Pat. No. 5,183,653; U.S. Pat. No. 5,387,409; and U.S.Pat. No. 5,118,797, the disclosures of which are incorporated byreference herein, in their entirety.

In another embodiment, disulfide bonds of a fibrin-binding polypeptideof the invention are used as two ligands for chelation of a radionuclidesuch as ⁹⁹mTc. In this way the peptide loop is expanded by theintroduction of Tc (peptide-S—S-peptide changed topeptide-S—Tc—S-peptide). This has also been used in other disulfidecontaining peptides in the literature (Chen et al., J. Nucl. Med.,42:1847-1855 (2001)) while maintaining biological activity. The otherchelating groups for Tc can be supplied by amide nitrogens of thebackbone, another cystine amino acid or other modifications of aminoacids.

Particularly preferred metal chelators include those of Formula 1, 2,and 3 (for ¹¹¹In and lanthanides such as paramagnetic Gd³⁺ andradioactive lanthanides, such as, for example ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, and¹⁶⁶Ho) and those of Formula 4, 5, and 6 (for radioactive ^(99m)Tc,¹⁸⁶Re, and ¹⁸⁸Re) set forth below.

These and other metal chelating groups are described in U.S. Pat. Nos.6,093,382 and 5,608,110, which are incorporated by reference herein intheir entirety. Additionally, the chelating group of Formula 3 isdescribed in, for example, U.S. Pat. No. 6,143,274; the chelating groupof Formula 5 is described in, for example, U.S. Pat. Nos. 5,627,286 and6,093,382, and the chelating group of Formula 6 is described in, forexample, U.S. Pat. Nos. 5,662,885; 5,780,006; and 5,976,495.

In the above Formulas 1 and 2, R is alkyl, preferably methyl. In theabove Formula 5, X is either CH₂ or O, Y is either C₁-C₁₀ branched orunbranched alkyl; Y is aryl, aryloxy, arylamino, arylaminoacyl; Y isarylkyl—where the alkyl group or groups attached to the aryl group areC₁-C₁₀ branched or unbranched alkyl groups, C₁-C₁₀ branched orunbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl orpolyhydroxy-polyalkoxyalkyl groups, J is C(═O)—, OC(═O)—, SO₂—, NC(═O)—,NC(═S)—, N(Y), NC(═NCH₃)—, NC(═NH)—, N═N—, homopolyamides orheteropolyamines derived from synthetic or naturally occurring aminoacids; all where n is 1-100. Other variants of these structures aredescribed, for example, in U.S. Pat. No. 6,093,382. The disclosures ofeach of the foregoing patents, applications and references areincorporated by reference herein, in their entirety.

Chelators may be covalently linked directly to the fibrin-binding moietyor linked to the fibrin-binding polypeptide via a linker, as describedpreviously, and then directly labeled with the radioactive metal ofchoice (see, WO 98/52618, U.S. Pat. No. 5,879,658, and U.S. Pat. No.5,849,261).

The selection of a proper radionuclide for use in a particularradiotherapeutic application depends on many factors, including:

-   -   a. Physical half-life—This should be long enough to allow        synthesis and purification of the radiotherapeutic construct        from radiometal and conjugate, and delivery of said construct to        the site of injection, without significant radioactive decay        prior to injection. Preferably, the radionuclide should have a        physical half-life between about 0.5 and 8 days.    -   b. Energy of the emission(s) from the radionuclide—Radionuclides        that are particle emitters (such as alpha emitters, beta        emitters and Auger electron emitters) are particularly useful,        as they emit highly energetic particles that deposit their        energy over short distances, thereby producing highly localized        damage. Beta emitting radionuclides are particularly preferred,        as the energy from beta particle emissions from these isotopes        is deposited within 5 to about 150 cell diameters.        Radiotherapeutic agents prepared from these nuclides are capable        of killing diseased cells that are relatively close to their        site of localization, but cannot travel long distances to damage        adjacent normal tissue such as bone marrow.    -   c. Specific activity (i.e. radioactivity per mass of the        radionuclide)—Radionuclides that have high specific activity        (e.g., generator produced ⁹⁰Y, ¹¹¹In, ¹⁷⁷Lu) are particularly        preferred. The specific activity of a radionuclide is determined        by its method of production, the particular target for which it        is produce, and the properties of the isotope in question.

Many of the lanthanides and lanthanoids include radioisotopes that havenuclear properties that make them suitable for use as radiotherapeuticagents, as they emit beta particles. Some of these are listed in thetable below.

TABLE 4 Approximate range Half-Life Max b- Gamma of b-particle (cellIsotope (days) energy (MeV) energy (keV) diameters) ⁴⁹-Pm 2.21 1.1 28660 ⁵³-Sm 1.93 0.69 103 30 ⁶⁶-Dy 3.40 0.40 82.5 15 ⁶⁶-Ho 1.12 1.8 80.6117 ⁷⁵-Yb 4.19 0.47 396 17 ⁷⁷-Lu 6.71 0.50 208 20 ⁰-Y 2.67 2.28 — 150¹¹-In 2.810 Auger electron 173, <5 * m emitter 247wherein: Pm is Promethium, Sm is Samarium, Dy is Dysprosium, Ho isHolmium, Yb is Ytterbium, Lu is Lutetium, Y is Yttrium, In is Indium.

The use of radioactive rhenium isotope as an alternative to abovelanthanides and lanthanoids is well known in the art and is encompassedby the invention.

Particularly ^(186/188)Re isotopes have proved to be of particularinterest in nuclear medicine, having a large number of applications inradiopharmaceutical therapy.

Thus, in a preferred embodiment, the invention includes novelradiotherapeutic agents in which fibrin-binding moieties of theinventions are conjugated to a suitably chelated radionuclide that emitsionizing radiations such as beta particles, alpha particles and Auger orCoster-Kroning electrons. More preferably, the fibrin binding moiety ofthe invention is labelled with a lanthanide or a lanthanoid radionuclideselected from ⁹⁰Y, ¹¹¹In, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, and ¹⁷⁷Lu.Examples of suitable chelating ligand may be selected from those ofFIGS. 6 a to 6 c.

The compounds of the invention labeled with therapeutic radionuclidescan find application either as a radiopharmaceutical that will be usedas a first line therapy in the treatment of a disease such as cancer, orin combination therapy, where the radiotherapeutic agents of theinvention could be utilized in conjunction with adjuvant chemotherapy(e.g, with one of the other therapeutic agents disclosed herein), or asthe therapeutic part of a matched pair therapeutic agent.

In fact, the peptide moiety of the radiotherapeutic of the invention isable to localize the chelated radioactive isotope to the pathologicfibrin deposition, for instance, into thrombi/clots, atheroscleroisplaques and inflammation-based damage involved in multiple sclerosisand, especially within solid tumors. The cytotoxic amount of ionizingradiation emitted by the localized radioisotope is thus able to causethe cell death of the pathologic tissue.

Complexes of radioactive technetium are particularly useful fordiagnostic imaging and complexes of radioactive rhenium are particularlyuseful for radiotherapy. In forming a complex of radioactive technetiumwith the reagents of this invention, the technetium complex, preferablya salt of Tc-99m pertechnetate, is reacted with the reagent in thepresence of a reducing agent. Preferred reducing agents are dithionite,stannous and ferrous ions; the most preferred reducing agent is stannouschloride. Means for preparing such complexes are conveniently providedin a kit form comprising a sealed vial containing a predeterminedquantity of a reagent of the invention to be labeled and a sufficientamount of reducing agent to label the reagent with Tc-99m.

Alternatively, the complex may be formed by reacting a peptide of thisinvention conjugated with an appropriate chelator with a pre-formedlabile complex of technetium and another compound known as a transferligand. This process is known as ligand exchange and is well known tothose skilled in the art. The labile complex may be formed using suchtransfer ligands as tartrate, citrate, gluconate or mannitol, forexample. Among the Tc-99m pertechnetate salts useful with the presentinvention are included the alkali metal salts such as the sodium salt,or ammonium salts or lower alkyl ammonium salts.

Preparation of the complexes of the present invention where the metal isradioactive rhenium may be accomplished using rhenium starting materialsin the +5 or +7 oxidation state. Examples of compounds in which rheniumis in the Re(VII) state are NH₄ReO₄ or KReO₄. Re(V) is available as, forexample, [ReOC₄](NBu₄), [ReOCl₄](AsPh4) ReOCl₃(PPh₃)₂ and asReO₂(pyridine)⁴⁺, where Ph is phenyl and Bu is n-butyl. Other rheniumreagents capable of forming a rhenium complex may also be used.

Radioactively-labeled scintigraphic imaging agents provided by thepresent invention are provided having a suitable amount ofradioactivity. In forming Tc-99m radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 mCi to 100 mCi permL.

Generally, the unit dose to be administered has a radioactivity of about0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution tobe injected at unit dosage is from about 0.01 mL to about 10 mL.

Typical doses of a radionuclide-labeled fibrin-binding imaging agentaccording to the invention provide 10-20 mCi for an adult human. Afterinjection of the fibrin-specific radionuclide imaging agent into thepatient, a gamma camera calibrated for the gamma ray energy of thenuclide incorporated in the imaging agent is used to image areas ofuptake of the agent and quantify the amount of radioactivity present inthe site. Imaging of the site in vivo can take place in a matter of afew minutes. However, imaging can take place, if desired, hours or evenlonger, after the radiolabeled peptide is injected into a patient. Inmost instances, a sufficient amount of the administered dose willaccumulate in the area to be imaged within about 0.1 of an hour topermit the taking of scintiphotos.

Proper dose schedules for the radiotherapeutic compounds of the presentinvention are known to those skilled in the art. The compounds can beadministered using many methods that include, but are not limited to, asingle or multiple IV or IP injections, using a quantity ofradioactivity that is sufficient to cause damage or ablation of thetargeted fibrin-containing tissue, but not so much that substantivedamage is caused to non-target (normal tissue). The quantity and doserequired is different for different constructs, depending on the energyand half-life of the isotope used, the degree of uptake and clearance ofthe agent from the body and the mass of the tumor. In general, doses canrange from a single dose of about 30-50 mCi to a cumulative dose of upto about 3 Curies for an adult human.

The radiotherapeutic compositions of the invention can includephysiologically acceptable buffers, and can require radiationstabilizers to prevent radiolytic damage to the compound prior toinjection. Radiation stabilizers are known to those skilled in the art,and may include, for example, para-aminobenzoic acid, ascorbic acid,gentistic acid and the like.

A single, or multi-vial kit that contains all of the components neededto prepare the complexes of this invention, other than the radionuclide,is an integral part of this invention.

A single-vial kit preferably contains a chelating ligand, a source ofstannous salt, or other pharmaceutically acceptable reducing agent, ifrequired, and is appropriately buffered with pharmaceutically acceptableacid or base to adjust the pH to a value of about 3 to about 9. Thequantity and type of reducing agent used would depend highly on thenature of the exchange complex to be formed. The proper conditions arewell known to those that are skilled in the art. It is preferred thatthe kit contents be in lyophilized form. Such a single vial kit mayoptionally contain labile or exchange ligands such as glucoheptonate,gluconate, mannitol, malate, citric or tartaric acid and can alsocontain reaction modifiers such as diethylenetriamine-pentaacetic acid(DPTA), ethylenediamine tetraacetic acid (EDTA), or α, β, or γcyclodextrin that serve to improve the radiochemical purity andstability of the final product. The kit may also contain stabilizers,bulking agents such as mannitol, that are designed to aid in thefreeze-drying process, and other additives known to those skilled in theart.

A multi-vial kit preferably contains the same general components butemploys more than one vial in reconstituting the radiopharmaceutical.For example, one vial may contain all of the ingredients that arerequired to form a labile Tc(V) complex on addition of pertechnetate(e.g., the stannous source or other reducing agent). Pertechnetate isadded to this vial, and after waiting an appropriate period of time, thecontents of this vial are added to a second vial that contains theligand, as well as buffers appropriate to adjust the pH to its optimalvalue. After a reaction time of about 5 to 60 minutes, the complexes ofthe present invention are formed. It is advantageous that the contentsof both vials of this multi-vial kit be lyophilized. As above, reactionmodifiers, exchange ligands, stabilizers, bulking agents, etc. may bepresent in either or both vials.

Therapeutic Applications

In another embodiment of the invention, a fibrin-binding moiety of theinvention is conjugated to a therapeutic agent (also referred to as atherapeutically active agent or moiety).

Unless otherwise provided, the term “therapeutic” as used hereinincludes at least partial alleviation of symptoms of a given condition.The therapeutically active agents do not have to produce a completealleviation of the symptoms to be useful. For example, treatment of anindividual can result in a decrease in the size of a tumor or diseasedarea or a blood clot, or even prevention of an increase in size of thetumor or diseased area, as well as partial alleviation of othersymptoms. Alternatively, treatment can also result in the reduction inthe number of blood vessels in an area of interest or can prevent anincrease in their number. Treatment can also prevent or lessen thenumber or size of metastatic outgrowths of the main tumor(s).

Suitable examples of therapeutic agents according to the presentinvention include anticoagulant-thrombolytic or fibrinolytic agentscapable of clots lysis, anti-angiogenic agents, cytotoxic agentsincluding chemotherapeutic or tumoricidal agents for selective killingand/or inhibiting the growth of cancer cells and, especially,radiotherapeutic agents.

In one embodiment the therapeutic agent is a thrombolytic orfibrinolytic agent. The fibrin-binding peptides of the present inventioncan be used to improve the activity of thrombolytic and anti-angiogenicagents by improving their affinity for fibrin and their residence timeat a fibrin clot or at a site of pathological angiogenic activity. Inthis aspect of the invention, hybrid thrombolytic agents are provided byconjugating a fibrin binding polypeptide according to the invention witha thrombolytic agent. Likewise, anti-angiogenic agents are provided byconjugating a fibrin-binding polypeptide according to the invention withan anti-angiogenic agent. The fibrin binding polypeptide portion of theconjugate causes the thrombolytic to “home” to the sites of fibrin clotsor sites of angiogenesis, and to improve the affinity of the conjugatefor such sites, so that the thrombolytic or anti-angiogenic activity ofthe conjugate is more localized and concentrated at the sites ofinterest.

Such conjugates will be especially useful in treating thrombusassociated diseases, especially acute myocardial infarction, stroke andpulmonary embolism in mammals, including humans, which method comprisesadministering to a mammal in need thereof an effective amount of afibrin binding moiety according to the invention conjugated with athrombolytic agent. The invention also provides the use of suchconjugates in the manufacture of a medicament for the treatment ofthrombus associated diseases in mammals, including humans. Suitablethrombolytic agents for use in this aspect of the invention includefibrinolytic enzymes, including plasminogen activators. The termplasminogen activator includes but is not limited to streptokinase,human tissue plasminogen activator (tPA) and urokinase (both single andtwo-chain forms). Such enzymes are obtained from natural sources ortissues or by recombinant production. Other suitable thrombolytic agentsinclude fibrinolytically active hybrid proteins (see, e.g., EP-A-155387) which comprise one chain of a 2-chain protease linked to a chain ofa different 2-chain protease, at least one of the chains in the hybridprotein being derived from a fibrinolytically active protease;thrombolytic protein conjugates (see, e.g., EP-A-152 736), such asurokinase linked to reversibly blocked plasmin; derivatives offibrinolytic enzymes in which the catalytic site on the enzyme which isresponsible for fibrinolytic activity is blocked by a human proteinattached thereto by way of a reversible linking group, for exampleurokinase reversibly linked to the active center of human plasmin;genetically engineered derivatives including muteins of naturallyoccurring plasminogen activators; hybrid molecules (see, e.g., EP-A-297882); reversibly blocked in vivo fibrinolytic enzymes, such as a binarycomplex between streptokinase and plasminogen, most preferably ap-anisoyl streptokinase/plasminogen complex without internal bondcleavage (anistreplase, described in U.S. Pat. No. 4,808,405); and thelike.

The thrombolytic agents and the fibrin binding moieties can be linked orfused in known ways, using the same type of linkers discussed above.Preferred linkers will be substituted or unsubstituted alkyl chains,amino acid chains, polyethylene glycol chains, and other simplepolymeric linkers known in the art. More preferably, if the thrombolyticagent is itself a protein, for which the encoding DNA sequence is known,the thrombolytic protein and fibrin binding polypeptide may becoexpressed from the same synthetic gene, created using recombinant DNAtechniques. The coding sequence for the fibrin binding polypeptide maybe fused in frame with that of the thrombolytic protein, such that thepeptide is expressed at the amino- or carboxy-terminus of thethrombolytic protein, or at a place between the termini, if it isdetermined that such placement would not destroy the required biologicalfunction of either the thrombolytic protein or fibrin bindingpolypeptide. A particular advantage of this general approach is thatconcatamerization of multiple, tandemly arranged fibrin bindingpolypeptides is possible, thereby increasing the number andconcentration of fibrin binding sites associated with each thrombolyticprotein. In this manner fibrin binding avidity is increased which wouldbe expected to improve the efficacy of the recombinant therapeuticprotein.

In addition to thrombolytic agents, the fibrin binding peptidesaccording to this invention can be used to deliver other active agentsto sites of fibrin in vivo or in vitro. For example, small moleculetherapeutics or other therapeutic agents may be linked to one or morefibrin binding peptides and the conjugate administered to a subject orintroduced to a fibrin-containing solution, and the fibrin-bindingproperties of the conjugate will concentrate the small molecule ortherapeutic agent at the sites of fibrin accumulation. In a particularlypreferred aspect, the fibrin binding peptides of the invention may beused to deliver agents which are active in the presence of fibrin, suchas angiogenesis promoters (e.g., fibroblast growth factor). The fibrinbinding peptides may also be used to increase the blood clearancehalf-life of a compound or drug, by causing accumulation of the compoundor drug in fibrin clots, from which it will be gradually released.

The fibrin-binding polypeptides of the present invention may also beused to target genetic material to specific cells. For example, thebinding peptides of the present invention may be used to localizegenetic material to cells or tissue containing fibrin. Thus suchconstructs may be useful in gene therapy. The genetic material mayinclude nucleic acids, such as RNA or DNA, of either natural orsynthetic origin, including recombinant RNA and DNA and antisense RNAand DNA. Types of genetic material that may be used include, forexample, genes carried on expression vectors such as plasmids,phagemids, cosmids, yeast artificial chromosomes (YACs) and defective or“helper” viruses, antigene nucleic acids, both single and doublestranded RNA and DNA and analogs thereof, such as phosphorothioate andphosphorodithioate oligodeoxynucleotides. Additionally, the geneticmaterial may be combined, for example, with lipids, proteins or otherpolymers. Delivery vehicles for genetic material may include, forexample, a virus particle, a retroviral or other gene therapy vector, aliposome, a complex of lipids (especially cationic lipids) and geneticmaterial, a complex of dextran derivatives and genetic material, etc.

In a preferred embodiment the binding polypeptides of the invention areutilized in gene therapy for treatment of diseases associated withangiogenesis. In this embodiment, genetic material, or one or moredelivery vehicles containing genetic material, e.g., useful in treatingan angiogenesis-related disease, may be conjugated to one or morefibrin-binding peptides of the invention and administered to a patient.

In the above treatment methods, the compounds may be administered by anyconvenient route customary for thrombolytic or therapeutic agents, forexample parenterally, enterally or intranasaly, and preferably byinfusion or bolus injection, or by depot or slow release formulation. Ina preferred embodiment, the composition may be formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anaesthetic such as lignocaine to ease pain at thesite of the injection. Generally, the ingredients will be suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent in activity units. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade “water for injection” or saline.Where the composition is to be administered by injection, an ampoule ofsterile water for injection or saline may be provided so that theingredients may be mixed prior to administration.

The quantity of material administered will depend on the seriousness ofthe thromboembolic condition and position and size of the clot. Theprecise dose to be employed and mode of administration must per force inview of the nature of the complaint be decided according to thecircumstances by the physician supervising treatment. In general,dosages of the fibrin binder/thrombolytic agent conjugate will followthe dosages that are routine for the thrombolytic agent alone, althoughthe improved affinity for fibrin added by the fibrin binder componentmay allow a decrease in the standard thrombolytic dosage. Particularthrombolytics contemplated for use in this therapy (with examples ofdose and method of administration) are as follows:

-   -   1 streptokinase 1.0-3.0 megaunits over 30 minutes to 3 hours        anistreplase 30 units; 2-5 minute injection tPA (wild-type)        50-150 mg; infusion over up to 6 hours two-chain urokinase        40-100 mg; infusion over up to 6 hours single-chain urokinase        (3-12 megaunits) 30-100 mg; infusion over up to 5 hours hybrid        plasminogen 20-100 mg; injection or infusion activators and        derivatives muteins of plasminogen 10-100 mg; injection or        infusion activators

In preferred features, the fibrin binding moiety is linked to thethrombolytic agent with a linker encompassing an enzymatic cleavagesite, e.g., an enzymatic cleavage site normally cleaved by enzymes inthe coagulation cascade, such as Factor Xa, thrombin, or plasmincleavage sites, etc. The thrombolytic agent preferably would not beactivated until it is cleaved from the fibrin binding moiety at the siteof the clot. Since cleavage of the thrombolytic agent would occur at thesite of the clot, the risk of unwanted bleeding events at sites distantfrom the clot would be minimized.

Alternatively, a therapeutic thrombolytic can be loaded into anultrasound vesicle that has been derivatized on its surface with thefibrin binding moieties of the present invention. The vesicle may alsobe filled with an ultrasound efficient gas, such as, but not limited to,perfluoropropane or perfluorobutane. Once the fibrin-specific vesiclehas homed to the site of a thrombus, as monitored by ultrasound, thefrequency and energy of the ultrasound waves administered can be alteredto result in a controlled release of the thrombolytic at the site of thethrombus (see, e.g., WO 93/25241).

As discussed above fibrin-binding peptides of the present invention alsocan be used to improve the activity of therapeutic agents such asanti-angiogenic or tumoricidal agents against undesired angiogenesissuch as occurs in neoplastic tumors, by homing in on areas undergoingangiogenesis so that the therapeutic activity can be more localized andconcentrated at the sites of angiogenesis.

In this aspect of the invention, hybrid agents are provided byconjugating a fibrin-binding polypeptide according to the invention witha therapeutic agent. The therapeutic agent may be a radiotherapeutic,discussed above, a drug, chemotherapeutic or tumoricidal agent, geneticmaterial or a gene delivery vehicle, etc. Such conjugates will be usefulin treating angiogenesis-associated diseases, especially neoplastictumor growth and metastasis, in mammals, including humans, which methodcomprises administering to a mammal in need thereof an effective amountof a fibrin-binding polypeptide according to the invention conjugatedwith a therapeutic agent. The invention also provides the use of suchconjugates in the manufacture of a medicament for the treatment ofangiogenesis associated diseases in mammals, including humans.

Suitable therapeutic agents for use in this aspect of the inventioninclude, but are not limited to: antineoplastic agents, such as platinumcompounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate,adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside,arabinosyl adenine, mercaptopolylysine, vincristine, busulfan,chlorambucil, melphalan (e.g., PAM, L-PAM, or phenylalanine mustard),mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin(actinomycin D), daunorubcin hydrochloride, doxorubicin hydrochloride,taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide,estramustine phosphate sodium, flutamide, leuprolide acetate, megestrolacetate, tamoxifen citrate, testoiactone, trilostane, amsacrine(m-AMSA), aparaginase (L-aparaginase), Erwina aparaginase, etoposide(VP-16), interferon cx-2a, Interferon cx-2b, teniposide (VM-26,vinblastine sulfate (VLB), vincristine sulfate, bleomycin sulfate,adriamycin, and arabinosyl; anti-angiogenic agents such as tyrosinekinase inhibitors with activity toward signaling molecules important inangiogenesis and/or tumor growth such as SU5416 and SU6668(Sugen/Pharmacia & Upjohn), endostatin (EntreMed), angiostatin(EntreMed), Combrestatin (Oxigene), cyclosporine, 5-fluorouracil,vinblastine, doxorubicin, paclitaxel, daunorubcin, immunotoxins;coagulation factors; antivirals such as acyclovir, amantadineazidothymidine (AZT or Zidovudine), ribavirin and vidarabine monohydrate(adenine arahinoside, ara-A); antibiotics, antimalarials, antiprotozoanssuch as chloroquine, hydroxychloroquine, metroidazole, quinine andmeglumine antimonate; anti-inflammatories such as diflunisal, ibuprofen,indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates.

As used herein the term “therapeutic” includes at least partialalleviation of symptoms of a given condition. The fibrin-bindingpeptides and conjugates of the present invention do not have to producea complete alleviation of symptoms to be useful. For example, treatmentof an individual can result in a decrease in the size of a tumor ordiseased area, or a blood clot or prevention of an increase in size ofthe tumor or diseased area or partial alleviation of other symptoms.Treatment can result in reduction in the number of blood vessels in anarea of interest or can prevent an increase in the number of bloodvessels in an area of interest. Treatment can also prevent or lessen thenumber or size of metastatic outgrowths of the main tumor(s).

Pharmaceutical Applications

Whether the fibrin binding moieties are to be used in patients fordetection and diagnosis or to facilitate the therapy, such uses requirethat they be treated as pharmaceutical agents. Pharmaceuticalcompositions of this invention comprise any of the compounds of thepresent invention, and pharmaceutically acceptable salts thereof, withany pharmaceutically acceptable ingredient, excipient, carrier, adjuvantor vehicle.

Pharmaceutical compositions of this invention can be administered tomammals including humans in a manner similar to other diagnostic ortherapeutic agents. The dosage to be administered, and the mode ofadministration will depend on a variety of factors including age,weight, sex, condition of the patient, and genetic factors, and willultimately be decided by the attending physician or veterinarian. Ingeneral, dosage required for diagnostic sensitivity or therapeuticefficacy will range from about 0.001 to 50,000 μg/kg, more usually 0.01to 25.0 μg/kg of host body mass.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

The pharmaceutical compositions of this invention may be administered bya variety of routes or modes. These include, but not limited, to oral,intratracheal, sublingual, pulmonary, topical, rectal, nasal, buccal,vaginal, parenteral, or via an implanted reservoir. Implanted reservoirsmay function by mechanical, osmotic, or other means. The term parenteralas used herein includes intraperitoneal, paravertebral, periarticular,periostal, subcutaneous, intracutaneous, intravenous, intra-arterial,intramuscular, intra-articular, intrasynovial, intrasternal,intrathecal, intralesional and intracranial injection or infusiontechniques.

Such compositions are preferably formulated for parenteraladministration, and most preferably for intravenous or intra-arterialadministration. Generally, and particularly when administration isintravenous or intra-arterial, pharmaceutical compositions may be givenas a bolus, as two or more doses separated in time, or as a constant ornon-linear flow infusion.

Details concerning dosages, dosage forms, modes of administration,composition and the like are further discussed in a standardpharmaceutical text, such as Remington's Pharmaceutical Sciences, 18thed., Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, Pa. 1990),which is hereby incorporated by reference.

As discussed supra, one embodiment of the present invention relates tonovel compounds comprising a fibrin-binding moiety conjugated with atleast one diagnostically or therapeutically active moiety.

In particular, a preferred embodiment of the present invention includescompounds of general Formula (I)A[-Y(-T)_(r)]_(s)  (I)

wherein

A is a fibrin-binding peptide moiety comprising an amino acid sequenceselected from the group consisting of the sequences provided in Table 1or Table 2;

Y is a suitable linking moiety connecting A with at least one T; when sis 2, the units Y may be the same or different from each other;

T is, independently in each occurrence, a diagnostically ortherapeutically active moiety;

s is 1 or 2,

r is, independently in each occurrence, an integer from 1 to 8;

or a physiologically acceptable salt thereof.

Unless otherwise specified, the phrases “fibrin-binding peptide moiety”or, simply, “peptide moiety,” used herein interchangeably, refer to asuitable derivative of the corresponding fibrin-binding peptide of thesequences disclosed herein, in which one or both of the N-terminal(—NH₂) and the C-terminal (—COOH) groups of the peptide arefunctionalized through formation of a carboxamido bond with Y. If theC-terminal or N-terminal group is not functionalized, it may be suitablyprotected or deactivated. Thus, “fibrin-binding peptide moiety” refersto that moiety resulting from the original amino acid sequence of thefibrin-binding peptide, following the said optionalprotection/deactivation and the said carboxamido bond(s) formation.

Typically, for instance, in the case of the fibrin-binding peptidehaving the amino acid sequence of Seq005 as shown in Table 1,H₂N—WQPCPAESWTFCWDP—COOH (SEQ ID NO. 1), the correspondingfibrin-binding peptide moieties include, for instance:

-HN-WQPCPAESWTFCWDP-CO-, Pg-HN-WQPCPAESWTFCWDP-CO-, and-HN-WQPCPAESWTFCWDP-CO-Pg

in which Pg is a suitable protecting/deactivating group.

In the present invention, unless otherwise indicated, the phrase“protecting group”, designates a protective group adapted to preservingthe function of the functional group to which it is bound. Specifically,protective groups are used to preserve amino function or carboxylfunction. Appropriate protective groups may include, for example,benzyl, benzyloxycarbonyl, alkyl or benzyl esters, or other substituentscommonly used for the protection of such functions, which are well knownto those skilled in the art, for example Fmoc, and protective groupsdescribed in conventional manuals such as T. W. Green, Protective Groupsin Organic Synthesis (Wiley, N.Y. 1981).

Unless otherwise indicated, the phrase “deactivating group” as usedherein, refers to chemical groups that are able to react with theN-terminal (—NH₂) or the C-terminal (—COOH) group of the peptide unittransforming it, through a chemical reaction, into a suitable derivativethereof that maintains the specificity of the corresponding peptidemoiety toward fibrin, but is unable to chemically react with,respectively, a (—COOH) or an (—NH₂) functionality on a differentmoiety, and thus may not be involved in carboxamido cross-linkingreaction.

Suitable examples of deactivating groups comprise Ac, where Ac isCH₃(CO)— when used, for instance, to deactivate an (—NH₂) terminal groupof the peptide chain to the corresponding, unreactive, AcHN— group. Onthe other side, —NH₂ or —NH(CH₃), may be, for instance, used todeactivate a terminal —COOH group by providing the corresponding —CONH₂or —CONH(CH₃) unreactive amide.

According to a preferred aspect of the invention, within the compoundsof Formula (I), A is a fibrin-binding peptide moiety comprising theamino acid sequence of Seq005 as shown in Table 1,H₂N—WQPCPAESWTFCWDP—COOH (SEQ ID NO.1), in which each of the W, Q, P, C,A, E, S, T, F and D has the meaning conventionally adopted when definingamino acids according to one letter code and in which the C amino acidsin positions 4 and 12 are bonded to each other through a disulfide(—S—S—) bond.

According to another preferred aspect of the invention, within thecompounds of Formula (I), s is 1. The compounds of the invention havings=1 include, independently in each occurrence, one or morediagnostically of therapeutically active moiety or moieties Tconjugated, through a suitable linking moiety Y, to the N-terminal(—NH₂) or, conversely, to the C-terminal (—COOH) group of the peptidemoiety, thus resulting in a compound of Formula (I) in which the peptidemoiety A is functionalized at only one of its N- or C-terminal groups.

According to another preferred aspect of the invention, within thecompounds of Formula (I), s is 2. The compounds of the invention inwhich s=2 include, independently in each occurrence, one or morediagnostically of therapeutically active moiety or moieties Tconjugated, through a suitable linking moiety Y, to each of theN-terminal (—NH₂) and the C-terminal (—COOH) groups of the peptidemoiety A, thus resulting in compounds of Formula (I) in which thepeptide moiety is functionalized at both of the N- and C-terminalgroups.

According to another preferred aspect of the invention, within thecompounds of Formula (I), r is an integer from 1 to 5.

In one preferred embodiment of the invention Y is a linear or brancheddivalent linking moiety. The phrase “divalent linking moiety” (or“divalent linker”, used herein interchangeably) is intended to include achain including a functional group which permits the conjugation of thelinking moiety with the N- or the C-terminal group of A and, a secondfunctional group which permits conjugation with a diagnostically or atherapeutically effective moiety T.

Unless otherwise indicated, the term “functional group” as used hereinrefers to specific groups of atoms within molecules or moieties that areresponsible for the characteristic chemical reactions of those moleculesor moieties. In the context of the invention, the functional groupsinclude the specific, suitably protected or suitably activated —NH₂terminal or —COOH terminal groups of the peptide moiety, of the linkingmoiety and of the diagnostically or therapeutically active moiety. Inaddition, these functional groups may include any other amine, thiol,carboxyl group present as a free group or as an optionally activatedreactive group on the said linking, diagnostically or therapeuticallyactive moiety, thus allowing other cross-linking or coupling reactions.In a preferred aspect of the present invention, the functional group aresuitably protected or suitably activated —NH₂ or —COOH groups allowingcross-linking reaction through carboxamido bonds (—NHCO—) and (—CONH—)formation.

Preferably, the divalent linking moiety is a linear C₁-C₁₀₀ and, morepreferably, a C₁-C₅₀, and, most preferably, a C₁-C₃₅ alkyl chain, thatis optionally interrupted by one or more groups selected from —O—,—CONH—, —CO—, —NR₁— and —NHCO—, and optionally substituted by one ormore —R₂ group(s), wherein R₁ is H or a C₁-C₅ alkyl group, and R₂ is a—CONH₂ group or a (C₁-C₅)alkyl group, that is optionally substituted, inits turn, by a —CONH₂ group or by an optionally substituted benzenering. As discussed supra, the said divalent linker includes twofunctional groups connecting it, respectively, with A and with T.

Unless otherwise provided, the term “(C₁-C₅) alkyl” as used herein,designates a linear or branched, saturated or unsaturated alkylsubstituent comprising from 1 to 5 carbon atoms such as, for example,methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl and thelike, wherein methyl, ethyl and propyl are preferred. More preferably,the divalent linking moiety Y includes one or more sub-units selected,for instance, from homobifunctional and heterobifunctional units andsuitable combinations thereof.

Unless otherwise provided, the term “homobifunctional” unit or moiety,refers to a unit or moiety having at least two reactive functionalgroups which are the same.

Unless otherwise provided, the term “heterobifunctional” unit or moiety,refers to a unit or moiety having at least two different reactivegroups.

Suitable examples of homobifunctional units include, for instance,dicarboxylic moieties and diamine moieties having formula, respectively,

—OC—Z—CO—, and

—NH—Z—NH—,

where Z is a chain preferably selected from the following:

—CH₂—(CH₂)_(n)—,

—CH₂—(CH₂O)_(m)—,

—(CH₂(CH₂)_(p)O)_(m)—(CH₂)_(m)—,

—(CH₂)_(n)—NHCO—(CH₂)_(n)—,

—(CH₂)_(n)—NHCO—CH₂O—(CH₂(CH₂)_(p)O)_(m)—(CH₂)_(m)—,

—(CH₂)_(p)—CH(R₂)—(CH₂)_(m)—,

—(CH₂)_(p)—CH(R₂)—(CH₂)_(m)—NHCO—CH₂O—(CH₂(CH₂)_(p)O)_(m)—(CH₂)_(m)—,

where n=1-10, m=1-5 and p=0-5, and derivatives thereof in which thecarboxylic and the amino group(s) are in a suitably activated orprotected form.

Suitable examples of heterobifunctional units, for instance, include:

—HN—(CH₂)_(n)—CO—,

HN—(CH₂)_(n)—CH(R₂)—CO—,

—HN—(CH₂)₂—O—(CH₂)₂—O—CH₂—CO—,

—HN—(CH₂)_(p)—CH(R₂)—(CH₂)_(m)—CO—,

—OC—CH(NR₁)—(CH₂)_(m)—NH—,

—OC—(CH₂)_(m)—NHOC—(CH₂)_(m)O—(CH₂)_(m)—NH—,

—HN—(CH₂)_(p)—CH(R₂)—(CH₂)_(m)—NHCO—CH₂O—(CH₂(CH₂)_(p)O)_(m)—(CH₂)_(m-)NH—,

wherein n, m and p are as above defined, and suitable combinationsthereof.

More preferably, the divalent linking moiety Y of the inventioncomprises one of the following units:

—HN—CH₂—CO—,

—OC—(CH₂)_(n)—CO—,

—HN—(CH₂)₂—O—(CH₂)₂—O—CH₂—CO—,

—HN—CH(CONH₂)—(CH₂)_(m)—NH—,

—NH—CH(CONH₂)—(CH₂)_(m)—NHCO—(CH₂)_(n)—CO—,

—CO—CH₂O—(CH₂)₂—O—(CH₂)₂—NHCO—CH₂O—(CH₂)₂—O—(CH₂)₂—NH—,

or a suitable repetition and/or combination thereof

In a particularly preferred aspect of the invention, the divalentlinking moiety Y is or comprises the unit -Gly-Gly-Gly-Lys in which Glyis Glycine and Lys is Lysine, also referred to as GGGK.

In a different embodiment of the invention, Y is a linear or branchedpolyfunctional linking moiety. Unless otherwise provided,“polyfunctional linking moiety”, and “polyfunctional linker”, usedherein interchangeably, refer to a linear or branched chain including atleast 3, preferably from 3 to 8 and, more preferably, from 3 to 5functional groups, one of them connecting the said polyfunctional moietywith the N-terminal (—NH₂) or the C-terminal group (—COOH) group of Aand the remaining connecting the polyfunctional moiety with at least 2,preferably, from 2 to 7, and, more preferably, from 2 to 5 equal ordifferent diagnostically or therapeutically effective moieties.

Preferably, the said polyfunctional linker Y is a linear or branchedC₁-C₁₅₀ and, preferably, a C₁-C₁₀₀ and, more preferably, a C₁-C₇₅ alkylchain, that is optionally interrupted by one or more groups selectedfrom —O—, —CONH—, —CO— —NHCO— and —NR₃, and optionally substituted byone or more —R₄ group(s), wherein R₃ is H or a C₁-C₅ alkyl groupoptionally substituted by a —COOH or —NH₂ group, and R₄ may be a groupselected from —NH₂, —COOH or a derivative thereof including, forinstance, lower alkyl esters or —CONH₂ amide, a (C₁-C₅)alkyl optionallysubstituted by a group selected from —COOH, —CONH₂ and —NHR₃ or by anoptionally substituted benzene ring, the chain further including atleast three functional groups connecting the polyfunctional moiety withA and each of the said remaining functional groups with a diagnosticallyor therapeutically effective moiety T.

Suitable examples of the said polyfunctional linking moiety may include,for instance:

-   -   (a) N-branched lysine systems (see, f. i., Veprek, P et al., J.        Pept. Sci. 5, 5 (1999); 5, 203 (1999),    -   (b) Polycarboxylic compounds and suitable derivative thereof in        which the carboxylic group(s) are in a suitably activated or        protected form,    -   (c) polyaminated compounds and suitable derivative thereof in        which the amino group(s) are in a suitably activated or        protected form,    -   (d) amino acids and poly-amino acids such as polyornithine,        polyarginine, polyglutamic acid, polyaspartic acid.

In a preferred aspect of the invention, the polyfunctional linkingmoiety Y includes one ore more sub-unit(s) selected from the abovehomobifunctional and heterobifunctional units and one or moresub-unit(s) selected from the following:

—HN—(CH₂)_(n)—CH(NR₃)—CO—,

—OC—CH(NR₃)—(CH₂)_(n)—NH—,

—OC—(CH₂)_(m)—NR₃—(CH₂)_(m)—CO—,

—HN—CH(R₄)—(CH₂)_(m)CO—,

—HN—CH(R₄)—(CH₂)_(n)—NH—,

—HN—(CH₂)_(p)—(CH₂)_(p)—CH(R₄))—(CH₂)_(m)—NH—,

—OC—(CH₂)_(p)—((CH₂)_(p)—CH(NR₃)—(CH₂)_(m)—NH—,

where n, m, p, R₃ and R₄ have the above defined meaning

In a particularly preferred aspect, the said polyfunctional Y moietycomprises one of the following sub-units:

-Gly-Gly-Gly-Lys,

—OC—(CH₂)_(p)—((CH₂)_(p)—CH(NH₂))—(CH₂)_(m)—NH—,

—HN—CH(CONH₂)—(CH₂)_(m)—NHCO—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—,

—OC—CH(NH₂)—(CH₂)_(m)—NHCO—CH(NH₂)—(CH₂)₂—NH—,

—OC—(CH₂)_(m)—CO—,

—HN—CH(COOH)—(CH₂)_(n)—NH—,

—HN—CH₂—CO—,

—OC—(CH₂)_(n)—CO—,

—HN—(CH₂)₂—O—(CH₂)₂—O—CH₂—CO—,

—OC—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—

or a suitable repetition and/or combination thereof in which each of theH₂N— and HOOC— groups of the sub-unit allows carboxamido cross-linkingreactions resulting in the possible elongation and/or ramification ofthe linking moiety.

Any intermediate compounds according to the invention including, forinstance, a peptide moiety A conjugated with a suitable, optionallyprotected Y moiety or with a suitable, optionally protected, sub-unit ofa Y moiety constitutes a further object of the present invention. Theseintermediates and their preparations are, for instance, detailed hereinbelow, in the experimental section.

As a non limiting example, FIG. 1 includes the synthetic procedure forthe preparation of a linker functionalized peptide moiety of theinvention (Seq005-P) in which the peptide moiety is the one comprisingthe amino acid sequence of Seq005 as shown in Table 1, and theconjugated linking moiety is -GGGKJJ- (SEQ ID NO. 139) (where J is theFmoc-8-amino-3,6-dioxaoctanoic acid; see Aldrich Neosystem and PeptidesInternational catalogue).

These novel intermediates including, the peptide moiety conjugated witha suitable linking unit or a suitable sub-unit thereof find applicationas intermediates for the preparation of the compounds of Formula (I).

According to a preferred aspect of the invention, within the compoundsof Formula (I), T is a diagnostically effective moiety or aradiotherapeutic moiety.

The phrase “diagnostically effective moiety” or “imaging effectivemoiety”, used herein interchangeably, refers to any moiety detectable byimaging diagnostic procedures, that is to say any moiety able toprovide, to improve or, in any way, to advantageously modify the signaldetected by an imaging diagnostic technique including, for instance,magnetic resonance imaging (MRI), radioimaging, x-ray imaging, lightimaging, thus enabling the registration of diagnostically useful,preferably contrasted, images when used in association with suchtechniques.

Examples of diagnostically effective moieties according to the inventioninclude, for instance, chelated gamma ray or positron emittingradionuclides; paramagnetic metal ions in the form of chelated orpolychelated complexes, X-ray absorbing agents including atoms of atomicnumber higher than 20; a dye molecule; a fluorescent molecule; aphosphorescent molecule; a molecule absorbing in the UV spectrum; aquantum dot; a molecule capable of absorption within near or farinfrared radiations; moieties detectable by ultrasound and, in general,all moieties which generate a detectable substance. The skilled personin the art well know that the imaging modality to be used have to beselected according to the imaging detectable moiety the diagnosticcompounds of the invention include.

MRI Contrast Agents

In a particularly preferred embodiment of the invention, within thecompound of Formula (I), T is an MRI detectable moiety.

Compounds of Formula (I) in which T is a MRI detectable moiety arepreferred for use as MRI contrast agents.

Accordingly, in one preferred aspect, the present invention relates tonovel MRI contrast agents of Formula (I) in which T is a MRI detectablemoiety.

Preferably, the said MRI detectable moiety comprises the residue of achelating ligand labelled with a paramagnetic metal element detectableby Magnetic Resonance Imaging (MRI) techniques.

Preferred paramagnetic metal elements are those having atomic numberranging between 20 and 31, 39, 42, 43, 44, 49 and between 57 and 83.More preferred are paramagnetic metal ions selected from the following:Fe(2+), Fe(3+), Cu(2+), Ni(2+), Rh(2+), Co(2+), Cr(3+), Gd(3+), Eu(3+),Dy(3+), Tb(3+), Pm(3+), Nd(3+), Tm(3+), Ce(3+), Y(3+), Ho(3+), Er(3+),La(3+), Yb(3+), Mn(3+), Mn(2+) wherein Gd(3+) is the most preferred.

The phrase “contrast imaging agent” or “contrast agent” refers to anydetectable entity that can be used to in vitro visualize or detectfibrin units or fibrin deposition into or on a biological elementincluding cells, biological fluids and biological tissues originatingfrom a live mammal patient, and preferably, from human patient, as wellas the in vivo identification and location of fibrin and fibrindeposition in or on mammalian and, preferably, human body organs,regions or tissues when the said detectable entity is used inassociation with a suitable diagnostic imaging technique.

The phrase “chelator”, “chelating ligand” or “chelating agent”, usedherein interchangeably, refers to chemical moieties, agents, compounds,or molecules characterized by the presence of polar groups able to aform a complex containing more than one coordinate bond with atransition metal or another metal entity. In a preferred aspect of theinvention the chelating ligands include cyclic or linear polyaminopolycarboxylic or phosphonic acids and contain at least one amine,thiol, carboxyl group, present as free, optionally activatedfunctionality that is suitable for use in the conjugation reaction witha functional group of the spacer chain Y.

The expression a “residue of a chelating agent”, or a “residue of achelating ligand”, used herein interchangeably, refers to that portionof the chelating ligand remaining after the above conjugation.Preferably the conjugation is from an acidic group on the chelatingligand or a suitable derivative thereof with an amino group (—NH2) ofthe linking moiety Y, or, alternatively, between a suitable reactiveamino group of the chelating ligand and a terminal carboxy group (—COOH)of the Y moiety, or a suitable derivative thereof, so as to give rise toa carboxamido linkage. The acidic or the reactive amino group of thechelating ligand involved in the crosslinking reaction is suitablyselected in order to not reduce or modify the chelating capability ofthe ligand residue.

The term “labelled” or “complexed” used in the context of a “chelatingligand labelled with a metal element”, refers to the formation of achelate or coordinate complex between the metal and the chelatingligand.

The term “metal entity” refers to a metal ion that is detectable by animaging technique. Such metal entities specifically include paramagneticmetal ions that are detectable by imaging techniques such as MagneticResonance Imaging (MRI), or to a metal ion (e.g. radionuclide) that isdetectable by imaging techniques such as scintigraphic imaging, SinglePhoton Emission Computed Tomography (SPECT) and Positron EmissionTomography (PET) or even a radionuclide for therapy.

Suitable chelating ligands include those discussed herein, particularlychelating ligands selected from the group consisting of: apolyaminopolycarboxylic acid and the derivative thereof, comprising, forexample, diethylenetriamine pentaacetic acid (DTPA) and derivativethereof such as benzo-DTPA, dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA,benzyl-DTPA, dibenzyl DTPA;N,N-bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]amino]ethyl]-glycine(DTPA-BMA);N-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl)]-N-[2-[bis(carboxymethyl)amino]ethylglycine(EOB-DTPA);4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oicacid (BOPTA);N,N-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamicacid 1-(1,1-dimethylethyl) esterN,N-bis[2-[bis(carboxymethyl)amino]ethyl]L-glutamic acid (DTPA-GLU);DTPA conjugated with Lys (DTPA-Lys); ethylenediaminetetraacetic acid(EDTA); 1,4,7,10-teraazacyclododecane 1,4,7,-triacetic acid (DO3A);1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA);[10-(2-hydroxypropyl)-1,4,7,10-teraazacyclododecane 1,4,7,-triaceticacid (HPDO3A);6-[bis(carboxymethyl)amino]tetrahydro-6-methyl-1H-1,4-diazepine-1,4(5H)-diaceticacid (AAZTA) provided by WO03008390 application, incorporated herein byreference, and derivative thereof;2-methyl-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid(MCTA);(α,α′,α″,α′″)-tetramethyl-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraaceticacid (DOTMA); or a polyaminophosphate acid ligand or derivative thereof,in particular,N,N′-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N′-diacetic acid(DPDP), ethylenedinitrilotetrakis(methylphosphonic) acid (EDTP) or apolyaminophosphonic acid ligand and derivative thereof, orpolyaminophosphinic acid and derivative thereof, in particular1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetrakis[methylphosphonic)]and1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetrakis[methylene-(methylphosphinic)]acid; or of macrocyclic chelators such as texaphyrins, porphyrins,phthalocyanines

Preferred ligand according to the present invention include those ofFIGS. 6 a to 6 c, which also include suitable bibliographic referencesconcerning their preparation.

Particularly preferred are: DTPA, DTPA-GLU, DTPA-Lys, DOTA, AAZTA, andthe following derivatives thereof:

Examples of particularly preferred MRI contrast agents of the inventioncomprise:

Nuclear Imaging (Radionuclide Imaging) and Radiotherapy

In another preferred embodiment of the invention, within the compoundsof Formula (I), T is a radioimaging detectable moiety or aradiotherapeutic moiety.

Compounds of Formula (I) in which T is a radio imaging detectable moietyare preferred for use as radiographic contrast agents.

Accordingly, in another preferred embodiment, the present inventionfurther relates to novel radiographic contrast agents of Formula (I) inwhich T is a radioimaging detectable moiety.

A “radioimaging detectable moiety” refers to a moiety that is detectableby imaging techniques such as scintigraphic imaging, Single PhotonEmission Computed Tomography (SPECT) and Positron Emission Tomography(PET).

Preferably, the radioimaging detectable moiety comprises the residue ofa chelating agent labelled with a radionuclide detectable by the saidscintigraphic, SPECT or PET imaging techniques.

When T is a radiotherapeutic moiety, it preferably comprises aradionuclide which is therapeutically effective. In a preferredembodiment, the radiotherapeutic moiety comprises the residue of achelating ligand labelled with a therapeutically active radionuclide.

Together with the chelating ligands discussed above, suitable examplesof chelating ligands for radionuclides may be selected from linear,macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelators (see, moreover,ligand disclosed, for instance, in U.S. Pat. No. 5,367,080, U.S. Pat.No. 5,364,613, U.S. Pat. No. 5,021,556, U.S. Pat. No. 5,075,099, U.S.Pat. No. 5,886,142), and other chelating ligands known in the artincluding, but not limited to, HYNIC, TETA and bisamino bisthiol (BAT)chelators (see also U.S. Pat. No. 5,720,934). For example, N₄ chelatingligands are described in U.S. Pat. Nos. 6,143,274; 6,093,382; 5,608,110;5,665,329; 5,656,254; and 5,688,487. Certain N₃S chelators are describedin PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. Nos.5,662,885; 5,976,495; and 5,780,006. The chelator may also includederivatives of the chelating ligandmercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N₃S, andN₂S₂ systems such as MAMA (monoamidemonoaminedithiols), DADS (N₂Sdiaminedithiols), CODADS and the like. These ligand systems and avariety of others are described in Liu and Edwards, Chem Rev, 1999, 99,2235-2268 and references therein.

The chelator may also include complexes containing ligand atoms that arenot donated to the metal in a tetradentate array. These include theboronic acid adducts of technetium and rhenium dioximes, such as aredescribed in U.S. Pat. Nos. 5,183,653; 5,387,409; and 5,118,797, thedisclosures of which are incorporated by reference herein, in theirentirety.

Preferred radionuclides according to the present invention include^(99m)Tc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹In, ¹¹³In, ¹⁶⁸Yb,¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu,⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi,¹⁰⁵Rh, ¹⁰⁹Pd, ^(117m)Sn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au, ¹¹¹Ag, ¹⁹⁹Au, ⁵¹Mn,^(52m)Mn, ⁵²Fe, ⁶⁰Cu, ⁷²As, ^(94m)Tc, ¹¹⁰In, ¹⁴²Pr, and ¹⁵⁹Ga.

The choice of a suitable ligand residue depends on the radionuclide usedfor the ligand labelling. Thus, preferred residues of chelating ligandsinclude those of FIGS. 6 a to 6 c (for ¹¹¹In lanthanides and radioactivelanthanides, including, for instance ¹⁷⁷Lu, ⁹⁰Y, ¹⁵³Sm, and ¹⁶⁶Ho or for⁶⁷Ga, ⁶⁸Ga, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, or ⁶⁷Cu) and those of FIGS. 7 a to 7 b(for radioactive ^(99m)Tc, ¹⁸⁶Re, and ¹⁸⁸Re). In particular, for metalentities including ¹¹¹In, lanthanides and radioactive lanthanides,particularly preferred are the following ligand residues

In the above Formulas 7 and 8, R is an alkyl, preferably methyl.For ^(99m)Tc, ¹⁸⁶Re, and ¹⁸⁸Re radionuclides, particularly preferred arethe following ligands:

as well as the following ligand residues:

These and other metal chelating groups are described in U.S. Pat. Nos.6,093,382 and 5,608,110, U.S. Pat. No. 6,143,274; U.S. Pat. Nos.5,627,286 and 6,093,382, U.S. Pat. Nos. 5,662,885; 5,780,006; and5,976,495 which are incorporated by reference herein in their entirety.Additionally, the above chelating group of Formula 9 is described in,for example, U.S. Pat. No. 6,143,274; the chelating groups of the aboveFormula 14 and 15 are described in U.S. Pat. Nos. 5,627,286 and6,093,382, and the chelating group of Formula 16 is described in, forexample, U.S. Pat. Nos. 5,662,885; 5,780,006; and 5,976,495.

In the Formula 14 and 15, X is either CH₂ or O, Y is either C₁-C₁₀branched or unbranched alkyl; Y is aryl, aryloxy, arylamino,arylaminoacyl; Y is arylkyl where the alkyl group or groups attached tothe aryl group are C₁-C₁₀ branched or unbranched alkyl groups, C₁-C₁₀branched or unbranched hydroxy or polyhydroxyalkyl groups orpolyalkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups, J is C(═O)—,OC(═O)—, SO₂, NC(═O)—, NC(═S)—, N(Y), NC(═NCH₃)—, NC(═NH)—, N═N—,homopolyamides or heteropolyamines derived from synthetic or naturallyoccurring amino acids; all where n is 1-100. Other variants of thesestructures are described, for example, in U.S. Pat. No. 6,093,382. Thedisclosures of each of the foregoing patents, applications andreferences are incorporated by reference herein, in their entirety.

The choice of the radionuclide will be determined based on the desiredtherapeutic or diagnostic application. For uses in radiotherapy orradiodiagnostics preferred radionuclides are ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As,¹¹¹In, ¹¹³In, ⁹⁰Y, ⁹⁷Ru, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ^(52m)Mn, ¹⁴⁰La, ¹⁷⁵Yb,¹⁵³Sm, ¹⁶⁶Ho, ¹⁴⁹Pm, ¹⁷⁷Lu, ¹⁴²Pr, ¹⁵⁹Gd, ²¹²Bi, ⁴⁷Sc, ¹⁴⁹Pm, ⁶⁷Cu,¹¹¹Ag, ¹⁹⁹Au, ¹⁶¹Tb, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹⁶⁸Yb, ⁸⁸Y, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁹⁷Ru,¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ^(99m)Tc, ²¹¹Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd,^(117m)Sn, ¹⁷⁷Sn and ¹⁹⁹Au and oxides and nitrides thereof. For example,for therapeutic purposes (e.g., to provide radiotherapy for primarytumors and metastasis), the preferred radionuclides include ⁶⁴Cu, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb,¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au, with ¹⁷⁷Lu and ⁹⁰Y being particularlypreferred. For diagnostic purposes (e.g., to diagnose and monitortherapeutic progress in e.g. primary tumors and metastases) thepreferred radionuclides include ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, and ¹¹¹In.

^(99m)Tc is particularly preferred for diagnostic applications becauseof its low cost, availability, imaging properties, and high specificactivity. The nuclear and radioactive properties of ^(99m)Tc make thisisotope an ideal scintigraphic imaging agent. This isotope has a singlephoton energy of 140 keV and a radioactive half-life of about 6 hours,and is readily available from a ⁹⁹Mo—^(99m)Tc generator.

Preferred metal radionuclides for use in PET imaging are positronemitting metal ions, such as ⁵¹Mn, ⁵²Fe, ⁶⁰Cu, ⁶⁸Ga, ⁷²As, ^(94m)Tc, or¹¹⁰In.

Preferred for scintigraphic applications are radiodiagnostic contrastagents of Formula (I) wherein T is the residue of a chelating ligand ofFIGS. 7 a to 7 b, labelled with radionuclide selected from ^(99m)Tc and^(186/188)Re. More preferred are those in which T is the residue of achelating ligand of formula from 22 to 33. Particularly preferred forscintigraphic applications are contrast agents of Formula (I) wherein Tis a residue of a ligands of formula from 22 to 33 labelled with^(99m)Tc.

Preferred radionuclides for use in radiotherapy include ⁶⁴Cu, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹In, ¹¹⁷Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ⁹⁰Y,^(186/188)Re, and ¹⁹⁹Au, with ¹⁷⁷Lu and ⁹⁰Y being particularlypreferred.

PET Imaging

In a still further embodiment of the invention, within the compounds ofFormula (I), T is an optionally labelled sugar moiety for use, whenlabelled, in PET Imaging.

Accordingly, in another preferred aspect, the present invention relatesto compounds of Formula (I) in which T is a suitably labelled sugarmoiety.

In a preferred embodiment of the invention, T includes a sugar moietylabeled by halogenation with radionuclides, such as, ¹²⁴I, ¹²⁵I, ¹³¹I,¹²³I, ⁷⁷Br, ⁷⁶Br and ¹⁸F, wherein ¹⁸F is particularly preferred.

Therapeutically Effective Agents

In another embodiment of the invention, within the compounds of Formula(I), T is a therapeutic active moiety.

Compounds of Formula (I) in which T is a therapeutic active moiety arenew and constitute a further object of the present invention.

Suitable examples of therapeutic active moieties according to thepresent invention include thrombolytic or fibrinolytic agents capable oflysis of clots, or cytotoxic agents for selective killing and/orinhibiting the growth of, for example, cancer cells, andradiotherapeutic agents.

In one embodiment of the invention T is one of the aforementionedthrombolytic or fibrinolytic agents and, preferably, streptokinase orurokinase. Compounds of Formula (I) in which T includes a thrombolyticor a fibrinolytic agent are useful in treating thrombus associateddiseases, especially acute myocardial infarction, in mammals, includinghumans.

Thus, in a different aspect thereof, the invention relates to the use ofa compound of Formula (I) in which T includes a thrombolytic or afibrinolytic agent for the preparation of a pharmaceutical formulationfor treating thrombus associated diseases in mammalian, includinghumans.

Thus, in a still different aspect thereof, the present invention relatesto compounds of Formula (I) in which T is an antineoplastic agent.

Suitable examples of the said agents include, for instance, thepreviously listed antineoplastic compounds as well as toxins.

In a preferred embodiment of the invention, T is a radiotherapeuticagent comprising a therapeutic radionuclide. Preferably, T is residue ofa chelating ligand that is labelled with a therapeutically activeradionuclide. These compounds are preferred for use as radiotherapeuticagents according to the invention.

Accordingly, in another embodiment, the invention relates to novelradiotherapeutic agents of Formula (I) in which T is the residue of achelating ligand suitably labelled with a therapeutically activeradionuclide.

Preferred radionuclides for use in radiotherapy include ⁶⁴Cu, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ⁹⁰Y,¹⁷⁷Lu, ^(186/188)Re, and ¹⁹⁹Au, with ¹⁷⁷Lu and ⁹⁰Y being particularlypreferred.

The selection of a proper radionuclide for use in a particularradiotherapeutic application depends on many factors, including:

-   -   a. Physical half-life—This should be long enough to allow        synthesis and purification of the radiotherapeutic construct        from radiometal and conjugate, and delivery of said construct to        the site of injection, without significant radioactive decay        prior to injection. Preferably, the radionuclide should have a        physical half-life between about 0.5 and 8 days.    -   b. Energy of the emission(s) from the radionuclide—Radionuclides        that are particle emitters (such as alpha emitters, beta        emitters and Auger electron emitters) are particularly useful,        as they emit highly energetic particles that deposit their        energy over short distances, thereby producing highly localized        damage. Beta emitting radionuclides are particularly preferred,        as the energy from beta particle emissions from these isotopes        is deposited within 5 to about 150 cell diameters.        Radiotherapeutic agents prepared from these nuclides are capable        of killing diseased cells that are relatively close to their        site of localization, but cannot travel long distances to damage        adjacent normal tissue such as bone marrow.    -   c. Specific activity (i.e. radioactivity per mass of the        radionuclide)—Radionuclides that have high specific activity        (e.g., generator produced ⁹⁰Y, ¹¹¹In, ¹⁷⁷Lu) are particularly        preferred. The specific activity of a radionuclide is determined        by its method of production, the particular target for which it        is produce, and the properties of the isotope in question.

Many of the lanthanides and lanthanoids include radioisotopes that havenuclear properties that make them suitable for use as radiotherapeuticagents, as they emit beta particles. Some of these are listed in thetable below.

TABLE NO. 4 Gamma Approximate range of Half-Life Max b-energy energyb-particle (cell Isotope (days) (MeV) (keV) diameters) ⁴⁹-Pm 2.21 1.1286 60 ⁵³-Sm 1.93 0.69 103 30 ⁶⁶-Dy 3.40 0.40 82.5 15 ⁶⁶-Ho 1.12 1.880.6 117 ⁷⁵-Yb 4.19 0.47 396 17 ⁷⁷-Lu 6.71 0.50 208 20 ⁰-Y 2.67 2.28 —150 ¹¹-In 2.810 Auger electron 173, <5 * m emitter 247wherein: Pm is Promethium, Sm is Samarium, Dy is Dysprosium, Ho isHolmium, Yb is Ytterbium, Lu is Lutetium, Y is Yttrium, In is Indium.

The use of radioactive rhenium isotope as an alternative to abovelanthanides and lanthanoids is well known in the art and is encompassedby the invention.

Particularly ^(186/188)Re isotopes have proved to be of particularinterest in nuclear medicine, having a large number of applications inradiopharmaceutical therapy.

Thus, in a preferred embodiment, the invention relates to novelradiotherapeutic agents of Formula (I) wherein T is the residue of asuitably chelated radionuclide that emits ionizing radiations such asbeta particles, alpha particles and Auger or Coster-Kroning electrons.

More preferably, T is the residue of a chelating ligand labelled with alanthanide or a lanthanoid radionuclide selected from ⁹⁰Y, ¹¹In, ¹⁴⁹Pm,¹⁵³Sm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, and ¹⁷⁷Lu. Examples of suitable chelatingligand may be selected from those of FIGS. 6 a to 6 c.

Thus, in a particularly preferred embodiment, the present inventionrelates to novel radiotherapeutic agent of Formula (I) wherein T is theresidue of a chelating ligand of FIGS. 6 a to 6 c, labelled with atherapeutically active nuclide ⁹⁰Y, ¹¹¹In or ¹⁷⁷Lu.

In another preferred aspect, the invention relates to novelradiotherapeutic agents of Formula (I) wherein T is the residue of achelating ligand of Formula 10 to 16 labelled with ¹⁸⁶Re or ¹⁸⁸Re.

The compounds of the invention labeled with therapeutic radionuclidescan find application either as radiopharmaceutical that will be used asa first line therapy in the treatment of a disease such as cancer, or incombination therapy, where the radiotherapeutic agents of the inventioncould be utilized in conjunction with adjuvant chemotherapy (e.g, withone of the other therapeutic agents disclosed herein), or as thetherapeutic part of a matched pair therapeutic agent.

In fact, the peptide moiety of the radiotherapeutic of the invention isable to localize the chelated radioactive isotope to the pathologicfibrin deposition, for instance, into thrombi/clots, atheroscleroisplaques and inflammation-based damage involved in multiple sclerosisand, especially within solid tumors. The cytotoxic amount of ionizingradiation emitted by the localized radioisotope is thus able to causethe cell death of the pathologic tissue.

Salts

Both the ligands and the paramagnetic or radionuclide chelated compoundsof Formula (I) can also be in the form of a physiologically salt.

The term “pharmaceutically acceptable salt”, as used herein, refers toderivatives of the compounds of the invention wherein the parentcompound is modified by making the acid or basic groups not yetinternally neutralized in the form of non-toxic, stable salts which doesnot destroy the pharmacological activity of the parent compound.

Suitable example of the said salts include: mineral or organic acidsalts, of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like.

Preferred cations of inorganic bases which can be suitably used toprepare salts within the invention comprise ions of alkali oralkaline-earth metals such as potassium, sodium, calcium or magnesium.Preferred cations of organic bases comprise, inter alia, those ofprimary, secondary and tertiary amines such as ethanolamine,diethanolamine, morpholine, glucamine, N-methylglucamine,N,N-dimethylglucamine.

Preferred anions of inorganic acids which can be suitably used to salifythe complexes of the invention comprise the ions of halo acids such aschlorides, bromides, iodides or other ions such as sulfate.

Preferred anions of organic acids comprise those of the acids routinelyused in pharmaceutical techniques for the salification of basicsubstances such as, for instance, acetate, succinate, citrate, fumarate,maleate or oxalate.

Preferred cations and anions of amino acids comprise, for example, thoseof taurine, glycine, lysine, arginine, ornithine or of aspartic andglutamic acids.

Optical Imaging

In one further preferred embodiment of the invention, T represents anoptically active imaging moiety.

Compounds of Formula (I) in which T is an optically active imagingmoiety are new and constitute a further object of the present invention.These compounds are preferred for use as for optical imaging contrastagents.

Thus, in a still further embodiment, the invention relates to novelcontrast agents for optical imaging having Formula (I), in which T is anoptically active imaging moiety.

Suitable examples of optically active imaging moieties include thosediscussed herein, for instance, a dye molecule; a fluorescent moleculesuch as, for example, fluorescein; a phosphorescent molecule; a moleculeabsorbing in the UV spectrum; a quantum dot; or a molecule capable ofabsorption of near or far infrared radiations.

Optical parameters to be detected in the preparation of an image mayinclude, e.g., transmitted radiation, absorption, fluorescent orphosphorescent emission, light reflection, changes in absorbanceamplitude or maxima, and elastically scattered radiation. For example,biological tissue is relatively translucent to light in the nearinfrared (NIR) wavelength range of 650-1000 nm. NIR radiation canpenetrate tissue up to several centimetres, permitting the use of thediagnostic agents of the invention comprising a NIR moiety to imagetarget-containing tissue in vivo.

Near infrared dye may include, cyanine or indocyanine derivatives, suchas, for example, Cy5.5, IRDye800, indocyanine green (ICG), indocyaninegreen derivatives including the tetrasulfonic acid substitutedindocyanine green (TS-ICG), and combination thereof.

In another embodiment, the compounds of the invention may includephotolabels, such as optical dyes, including organic chromophores orfluorophores, having extensively conjugated and hence delocalized ringsystems and having absorption or emission maxima in the range of400-1500 nm. The compounds of the invention may alternatively bederivatized with a bioluminescent molecule. The preferred range ofabsorption maxima for photolabels is between 600 and 1000 nm to minimizeinterference with the signal from hemoglobin. Preferably,photoabsorption labels have large molar absorptivities, e.g. >10⁵cm⁻¹M⁻¹, while fluorescent optical dyes will have high quantum yields.Examples of optical dyes include, but are not limited, to thosedescribed in WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO 98/47538, andreferences cited therein.

For example, the photolabels may be covalently linked directly to thepeptide moiety of the invention, or one or more of them may be linkedthereto through a branching chain Y, as described previously.

After injection of the optically-labeled diagnostic derivative accordingto the invention, the patient is scanned with one or more light sources(e.g., a laser) in the wavelength range appropriate for the photolabelemployed in the agent. The light used may be monochromatic orpolychromatic and continuous or pulsed. Transmitted, scattered, orreflected light is detected via a photodetector tuned to one or multiplewavelengths to determine the location of target-containing tissue (e.g.angiogenic tissue) in the subject. Changes in the optical parameter maybe monitored over time to detect accumulation of the optically-labeledreagent at the target site. Standard image processing and detectingdevices may be used in conjunction with the optical imaging reagents ofthe present invention.

In a preferred embodiment, the invention relates to novel opticalimaging agents of Formula (I) wherein T is the residue of a5-Carboxyfluorescein.

Use of Compounds of Formula I for Imaging and Treating PathologicalConditions Associated with Fibrin, Particularly Tumors

As discussed above, the peptide moiety compounds of the invention areable to selectively bind to fibrin and, in particular, to fibrin presentin the extracellular matrix (EC) of tumor or connective tissue of stromathus acting as targeting moiety able to localize an active moiety linkedthereto to fibrin depositions and, especially, to fibrin depositioninside solid tumors or metastatic tissues.

The compounds of the present invention may thus find advantageousapplication for the diagnosis, prevention and treatment of allpathological conditions associated with fibrin deposition, includingclots and thromboembolic diseases and, especially, solid tumors andmetastatic processes. Moreover, they may advantageously be used tofollow up and monitor oncological therapy efficacy and tumor treatmentresults.

In particular, the compounds of Formula (I) in which T is adiagnostically active moiety according to the invention may findadvantageous application for localizing and diagnostically visualizingfibrin deposition associated with atherosclerosis and plaque formation.In a further aspect compounds of the invention allow diagnostic imagingof inflammatory processes, including demyelination processes and axonaldamage involved in multiple sclerosis and, in general, of allinflammatory conditions associated with processes in which fibrin playsa role. In an especially preferred aspect, the compounds of Formula (I)in which T is a diagnostically active moiety according to the inventionmay find advantageous application in localizing and visualizing fibrincontent inside solid tumor and metastatic processes.

In a further aspect, the compounds of Formula (I) in which T is adiagnostically active moiety may find application in the non invasivehistopathologic grading of solid tumors. A correlation, in fact, existsbetween the MRI derived measures of the fibrin content inside the tumormass the compounds of the invention provide and the histopathologicgrade of the said solid tumor.

The compounds of Formula (I) in which T is a therapeutically activemoiety may find advantageous application for prevention, ameliorationand/or treatment of pathological conditions associated with fibrindeposition, including clots and thromboembolic diseases andatherosclerotic plaques and inflammatory damages associated with fibrindeposition. In an especially preferred aspect, the compounds of Formula(I) in which T is a therapeutically active moiety may find advantageousapplication for preventing, ameliorating and/or treating solid tumorsand metastatic processes associated therewith. In an even more preferredembodiment, the compounds of Formula (I) in which T is aradiotherapeutic moiety may be advantageously used to provideradiotherapy to a patient (particularly a human) in need thereof due tothe presence of one or more solid tumors and metastatic processesassociated therewith.

In another embodiment, the invention concerns pharmaceuticalcompositions containing, as an active ingredient, at least one compoundof Formula (I), including pharmaceutically acceptable salts thereof, incombination with one or more possible pharmaceutically acceptablecarriers or excipients.

In an even further aspect thereof, the invention relates to the use ofthe compounds of Formula (I) in which T is a diagnostically activemoiety for the diagnosis in vitro (of ex vivo samples) of pathologicalsystems, including cells, biological fluids and biological tissuesoriginating from a live mammal patient, and preferably, human patient.Additionally, the invention relates to the use of the compounds ofFormula (I) in which T is a diagnostically active moiety for thepreparation of pharmaceutical compositions for use in the diagnosticimaging, in vivo, of human body organ, regions or tissues, includingtumorous or cancerous tissues and inflammations, wherein fibrindepositions occur.

In yet another aspect the invention provides a method for imaging solidtumors or tumorous cells both in vitro (of ex vivo samples) and in vivo,the method comprising the use of a diagnostic imaging agent of theinvention and an imaging technique.

Furthermore, the invention provides a method for treating and/orameliorating solid tumors or tumorous cells in vivo, the methodcomprising administering a therapeutic agent of the invention.

Preparations

The preparation of the compounds of Formula (I), in which T is theresidue of a chelating agent labelled with a paramagnetic metal ion or aradionuclide, either as such or in the form of physiologicallyacceptable salts, represents a further object of the invention.

Isolation, conjugation and use of fibrin binding moieties (andconjugates thereof with diagnostically or therapeutically activemoieties) in accordance with this invention will be further illustratedin the following examples. The specific parameters included in thefollowing examples are intended to illustrate the practice of theinvention, and they are not presented to in any way limit the scope ofthe invention.

EXAMPLES

The following materials were used in performing the Examples below:

Materials: Fmoc-protected amino acids used were obtained fromNova-Biochem (San Diego, Calif., USA), Advanced ChemTech (Louisville,Ky., USA), Chem-Impex International (Wood Dale Ill., USA), and MultiplePeptide Systems (San Diego, Calif., USA). DPPE, DSPE-PG4-NH₂, andDPPE-PG4-NH₂ were obtained from Avanti Polar Lipids (Alabaster, Ala.).Fmoc-PEG3400-NHS was obtained from Shearwater Polymers (Huntsville,Ala.). Other reagents were obtained from Aldrich Chemical Co.(Milwaukee, Wis.) and VWR Scientific Products (Bridgeport, N.J.).Solvents for peptide synthesis were obtained from Pharmco Co.(Brookfield, Conn.).

Examples 1-7 and 23 utilize and make reference to procedures A-Ldescribed below.

Procedures for Peptide Synthesis

Procedure A: Automated Peptide Solid Phase Peptide Synthesis

Individual peptides were prepared using an ABI 433A instrument (AppliedBiosystems, Foster City, Calif.). PAL-Peg-PS-Resin (1.2 g, 0.18 mmol/g)or NovaSyn TGR resin (1.25 g, 0.20 mmol/g) (NovaBiochem, Novato, Calif.)was used for all syntheses. The peptides were assembled on resin usingthe FastMoc™ protocol. After the synthesis, the resin was washed withDCM (2×) and dried.

Procedure B: Manual Coupling of Amino Acids

DMF was used as the coupling solvent unless otherwise stated. Theappropriate Fmoc-amino acid in DMF (0.25M solution, 3 equiv) was treatedwith HATU (0.5M in NMP, 3.0 equiv) and DIEA (6.0 equiv). The mixture wasshaken for ˜2 min and then was transferred to the synthesis vesselcontaining the resin. The vessel was then shaken overnight at ambienttemperature. The resin was filtered to remove excess reagents and thenwashed (4×) with DMF.

Procedure C: Manual Removal of the Fmoc Protecting Group

The resin containing the Fmoc-protected amino acid was treated with 20%piperidine in DMF (v/v, 15.0 mL/g resin) for 10 min. The solution wasdrained from the resin. This procedure was repeated once and thenfollowed by washing the resin with DMF (4×).

Procedure D: Removal of the ivDde Group (Solid Phase)

The resin containing the ivDde-protected amino acid was treated with 10%(v/v) hydrazine in DMF (10 mL/g resin) for 10 min. The solution wasdrained from the resin. This procedure was repeated once and thenfollowed by washing the resin with DMF (4×).

Procedure E: Removal of the ivDde Group from Peptides in Solution

The peptide (50 mg) was dissolved in DMF (2.0 mL) and treated with neathydrazine (40-200 μL) for 10 min. The mixture was diluted with water toa volume of 10 mL and this was directly applied to a C18 reverse phasecolumn and purified by preparative HPLC as described in the generalprocedures.

Procedure F: Coupling of Fmoc-Adoa (Fmoc-J)

Fmoc-Adoa (2 equiv) and HATU (2 equiv) were dissolved in DMF and DIEA (4equiv) was added to the mixture. The mixture was stirred for 1 minbefore transferring the activated acid to the resin. The concentrationof reagents was as discussed above for standard peptide couplings. Thecoupling was continued for 12 h at ambient temperature. The resin wasdrained of the reactants and washed with DMF (4×). In cases where twoAdoa units were appended to the resin, the Fmoc group of the firstappended Fmoc-Adoa unit was removed (procedure C), the resin washed withDMF (4×) and followed by coupling of the second Adoa moiety.

Procedure G: Cleavage and Side-Chain Deprotection of Resin BoundPeptides

Reagent B (88:5:5:2—TFA:water:phenol:TIPS—v/v/wt/v), 15 mL/g resin, wasadded to ˜1.0 g of the resin and the vessel was shaken for 4.5 h atambient temperature. The resin was filtered and washed twice with TFA (5mL/g resin). The filtrates were combined, concentrated to give a syrupwhich upon Trituration with 20 mL of Et₂O/g of resin gave a solidresidue which was stirred for 5-15 min and then centrifuged. Thesupernatant was decanted and the process was repeated three times. Theresulting solid was dried under high vacuum or with a stream of drynitrogen gas.

Procedure H: Disulfide Cyclization

The precipitate obtained from trituration of the crude cleavage mixturewith Et₂O was transferred to a beaker and DMSO (5-10 μL/mg crudepeptide) was added. The pH of the solution was adjusted to 8 by addingN-methyl-D-glucamine (10-100 mM in H₂O). The mixture was stirred for 48h and was then purified by preparative HPLC.

Procedure I: Preparation of 5-Carboxyfluorescein (CF5) Derivatives ofPeptides

To a solution of a peptide in DMF (15 μL/mg) and DIEA (20 equiv/equivpeptide) was added 5-carboxyfluorescein NHS ester (1.3-1.5 equiv.) inDMF (20 μL/mg). The mixture was stirred for 1-3 h. The reaction wasmonitored by mass spectroscopy and analytical HPLC. Upon completion ofthe reaction, the crude was filtered and purified by preparative HPLC.

Procedure J: Preparation of Aloc-Gly-OH

Gly-O-t-Bu.AcOH (1 g, 5.24 mmol) was dissolved in DCM (15 mL), anddiallyl pyrocarbonate (1.1 g, 5.91 mmol, 1.13 equiv) was added dropwise.The mixture was stirred at ambient temperature for 0.5 h. Then DIEA (3.7g, 5 mL, 28.68 mmol, 5.47 equiv) was added. The mixture was stirred atambient temperature overnight. The volatiles were removed and the cruderesidue was dissolved in EtOAc (100 mL/g of crude) and the organic layerwas washed with 1N HCl (2×). The volatiles were removed and the crudewas dried at high vacuum. NMR (500 MHz, CDCl₃) indicated a pure productand was consistent with the structure. The crude was then dissolved in asolution of TFA/DCM (1/1, v/v, 25 mL) and the solution was stirredovernight. The volatiles were removed, EtOAc was added to wash anyresidue from the wall of the flask and then the volatiles were removedon the rotary evaporator. This was repeated. The resulting product wasdried overnight at high vacuum. NMR spectroscopy of the material (CDCl₃,500 MHz) was consistent with the expected structure and the purity wasfound to be sufficient for use in manual coupling protocols.

Procedure K: Preparation of Aloc-Arg(Pmc)-OH

H-Arg(Pmc)-OH (5 g, 11.35 mmol) was dissolved in a mixture of H₂O andDioxane (1/1, v/v, 125 mL), and diallyl pyrocarbonate (6.34 g, 34.05mmol, 3.0 equiv) was added. The pH of the mixture was adjusted to >10.0by adding Na₂CO₃. The mixture was stirred and kept at reflux overnight.The volatiles were removed by rotary evaporation, the crude wasdissolved in EtOAc (100 mL/g of crude) and the solution was washed with1N HCl (2×). The volatiles were removed by rotary evaporation, the crudewas dissolved in CHCl₃ and the solution was loaded onto a silica gelcolumn. The column was eluted with two column volumes of CHCl₃ and thensimilarly eluted with a 5% solution of MeOH in CHCl₃. Fractionscontaining the desired compound were combined and the volatiles wereremoved by rotary evaporation and pumping at high vacuum to provide 4.2g (70% yield) of Aloc-Arg(Pmc)-OH. The proton NMR spectrum (CDCl₃, 500MHz) was consistent with the expected structure and required purity.

Procedure L: Removal of the Aloc Protecting Group from Peptides

The Aloc-protected peptide was dissolved in 5-20 mL/100 mg peptide of asolution of NMM:acetic acid:DMF (1:2:10). Pd(PPh₃)₄ (1-10 equiv/equivpeptide) was added. The mixture was stirred for 0.5-4 h. MS andanalytical HPLC were used to check the reaction. After the reaction wascomplete, the crude reaction mixture was diluted to twice its volumewith 10%-25% CH₃CN in H₂O, filtered and purified by preparative HPLC.

Methods for Analysis and Purification

Analytical HPLC

Column: Waters Corp. X-Terra, MS—C₁₈; 4.6 mm i.d.×50 mm; 5 μm particle;Eluent A: Water (HPLC Grade with 0.1% TFA by weight); Eluent B:Acetonitrile (0.1% TFA by weight). Initial conditions and gradientelution profiles employed are described in the respective experimentalprocedures for analysis of the title compounds. Elution rate: 3 mL/min;Detection: UV at 220 nm.

Preparative HPLC Purification

Column: Waters Corp. X-Terra MS—C₁₈; 50 mm i.d.×250 mm; 10 μm particle;Eluents: Eluent A: Water (HPLC Grade with 0.1% TFA by weight); Eluent B:Acetonitrile (0.1% TFA by weight); Initial conditions and gradientelution profiles employed are described in the respective experimentalprocedures for analysis of the title compounds. Elution rate: 100mL/min; Detection: UV at 220 nm.

Preparative HPLC Purification for Phospholipid Peptide Conjugates

Purification of Phospholipid Peptide Conjugates Employing the KromasilPrep C4 HPLC Column

The reaction mixture was diluted with distilled deionized water andpurified on a reverse phase C4 preparative column (Kromasil® Prep C₄,particle size 10 μm, pore size 300 Å, 20×250 mm), using a gradient of50-100% water (0.1% TFA) into CH₃OH:CH₃CN (1:1, v/v, 0.1% TFA) at 100mL/min over a period of 30 min. Fractions (15 mL) were analyzed by HPLC(column: YMC C-4, 5 μm, 300 Å, 4.6×250 mm) and the pureproduct-containing fractions were pooled. Methanol was removed from thecombined product-containing eluates by rotary evaporation; the resultingsolution was diluted with 10% aqueous acetonitrile, frozen andlyophilized to provide the desired product.

Purification of Phospholipid Peptide Conjugates Employing the ZorbaxPrep C-3 HPLC Column

The diluted reaction mixture was loaded onto a Zorbax C-3 column (21.2mm i.d.×150 mm) which was pre-equilibrated with 25% B (CH₃CN with 0.1%TFA) at a flow rate of 30 mL/min. The column was eluted at 30 mL/minwith the same eluent until the plug of DMF was eluted. The proportion ofeluent B was then increased from 25% B to 30% B over 3 min and thenramped to 100% B over 50 min. Fractions (15 mL) were collected andproduct-containing fractions were pooled, frozen and lyophilized.

HPLC Methods Employed for Analysis of Compounds

HPLC Systems Employed for Analysis of Peptides and Phospholipid-PeptideConjugates

System A: Column: Waters XTerra MS-C18 4.6×50 mm; Particle size: 5microns; Eluents: A: Water (0.1% TFA), B: Acetonitrile (0.1% TFA);Elution: linear gradient 5-55% B in 7 min; Flow rate: 3 mL/min;Detection: UV, λ=220 nm.

System B: Column: Waters XTerra MS-C18 4.6×50 mm; Particle size: 5microns; Eluents: A: Water (0.1% TFA), B: Acetonitrile (0.1% TFA);Elution: linear gradient 5-65% B in 7 min; Flow rate: 3 mL/min;Detection: UV, λ=220 nm.

System C: Column: Waters XTerra MS-C18 4.6×50 mm; Particle size: 5microns; Eluents: A: Water (0.1% TFA), B: Acetonitrile (0.1% TFA);Elution: linear gradient 15-65% B in 7 min; Flow rate: 3 mL/min;Detection: UV, λ=220 nm.

System D: Column: Waters XTerra MS-C18 4.6×50 mm; Particle size: 5microns; Eluents: A: Water (0.1% TFA), B: Acetonitrile (0.1% TFA);Elution: Isocratic at 15% B for 1 min, then linear gradient 15-70% B in6 min; Flow rate: 3 mL/min; Detection: UV, λ=220 nm.

System E: Column: Waters XTerra MS-C18, 4.6 mm i.d.×50 mm; Particlesize: 5 microns; Eluents: A: Water (0.1% TFA), B: Acetonitrile (0.1%TFA); Elution: linear gradient 15-60% B in 6 min; Flow rate: 3.0 mL/min;Detection: UV, λ=220 nm.

System F: Column: YMC C18, 4.6×250 mm; Eluents: A: Water (0.1% TFA), B:Acetonitrile (0.1% TFA), Initial condition: 20% B, Elution: lineargradient 20-80% B in 20 min; Flow rate: 1.0 mL/min; Detection: UV, λ=220nm.

System G: Column: Waters XTerra MS-C18, 4.6 mm i.d.×50 mm; Particlesize: 5 microns; Eluents: A: Water (0.1% TFA), B: acetonitrile (0.1%TFA); Elution: Initial condition: 10% B, linear gradient 10-50% B over 8min; Flow rate: 3 mL/min; Detection: UV, λ=220 nm.

System H: Column: Waters XTerra MS-C18 4.6×50 mm; Particle size: 5microns; Eluents: A: Water (0.1% TFA), B: Acetonitrile (0.1% TFA);Elution: Initial condition: 5% B, linear gradient 5-65% B in 8 min; Flowrate: 3 ml/min; Detection: UV @ 220 nm.

System I: Column: Waters XTerra C-4, 4.6×50 mm; Eluents: A: Water (0.1%TFA), B: Acetonitrile:Methanol (1:1)(0.1% TFA); Elution: Initialcondition: 80% B, linear gradient 80-100% B in 6 min.; Flow rate: 3.0mL/min; Detection: UV, λ=220 nm.

System J: Column: YMC C-4, 4.6×250 mm; Eluents: A: Water (0.1% TFA), B:Acetonitrile:Methanol (1:1)(0.1% TFA); Elution: Initial condition: 80%B, linear gradient 80-100% B in 50 min.; Flow rate: 2.0 mL/min;Detection: UV, λ=220 nm and ELSD: Sensitivity 10, Temp. 51 Deg C.,Pressure 2.2 Torr.

System K: Column: YMC C4; 250 mm×4.6 mm i.d.; Particle size: 5.0microns; Eluents: A:Water (0.1% TFA), B: acetonitrile (0.1% TFA);Elution: Initial condition: 80% B, linear gradient 80-90% B over 100min, then ramp to 100% B over 1 min, then hold at 100% B for 1 min; Flowrate: 2.0 mL/min; Detection: UV, λ=220 nm and ELSD: Sensitivity 10,Temp. 51 Deg C., Pressure 2.2 Torr.

System L: Column: YMC C-4, 4.6×250 mm; Eluents: A: Water (0.1% TFA), B:Acetonitrile/Methanol (1:1, v/v)(0.1% TFA); Elution: Initial condition:60% B, linear gradient 60-100% B in 20 min.; Flow rate: 2.0 mL/min;Detection: UV, λ=220 nm and ELSD: Sensitivity 10, Temp. 51 Deg C.,Pressure 2.2 Torr.

System M: Column: YMC C-4, 4.6×250 mm; Eluents: A: Water (0.1% TFA), B:Acetonitrile/Methanol (1:1, v/v)(0.1% TFA); Elution: Initial condition:50% B, linear gradient 50-90% B in 10 min.; Flow rate: 3.0 mL/min;Detection: UV, λ=220 nm and ELSD: Sensitivity 10, Temp. 51 Deg C.,Pressure 2.2 Torr.

System N: Column: Zorbax 300SB C-3, 3 mm i.d.×150 mm; 3.5 μm particle;Eluent A: Water (0.1% TFA); Eluent B: Acetonitrile (0.1% TFA). Initialcondition: 50% B; Elution:linear gradient 50-90% B over 3 min, hold at90% B for 11 min; Elution rate: 0.5 mL/min; Detection: UV, λ=220 nm andELSD: Sensitivity 10, Temp. 51 Deg C., Pressure 2.2 Ton.

System O: Column: YMC C-4, 4.6×50 mm; Eluents: A: Water (0.1% TFA), B:Acetonitrile-Methanol (1:1 v/v)(0.1% TFA); Elution: Initial condition:75% B, linear gradient 70-100% B in 10 min.; Flow rate: 3.0 mL/min;Detection: UV, λ=220 nm and ELSD: Sensitivity 10, Temp. 51 deg C.,Pressure 2.2 Torr.

System P: Column: ES industries MacroSep C4, 4.6×50 mm; Particle size: 5g; Eluents: A: Water (0.1% TFA), B: Acetonitrile/Methanol (1:1,v/v)(0.1% TFA); Elution: Initial condition: 25% B, linear gradient25-100% over 7 min; Flow rate; 3 mL/min; Detection: UV @ 220 nm

Assay of Fibrin Binding Peptides by Direct Binding FluorescencePolarization (FP) Procedure Using 5-Carboxyfluoresein Labeled Peptides

Protocol

-   -   1. Prepare 1 mL of 40 nM solution of        5-carboxyfluorescein-labeled peptide in HEPES dilution buffer        (HDB) with 0.01% Tween.        -   HDB (10 mM HEPES, 150 mM NaCl₂, 2 mM CaCl₂)    -   2. Dilute the 40 nM solution to obtain 1 ml of a 20 nM solution        of the test 5-carboxyfluorescein-labeled peptide.    -   3. Prepare a solution of DDE at a concentration that will be        approximately 5-10 fold greater than the expected K_(D). For the        described assay an 8 μM concentration of DDE was prepared.    -   4. Mix equal volumes of the DDE solution with the 40 nM peptide        solution.    -   5. Prepare serial dilutions of DDE in a solution consisting of        the binding buffer with 0.01% Tween20 and 20 nM of the        5-carboxyfluorescein-labeled peptide.    -   6. Make dilutions as shown in the following table in a        Labsystems 384-well microplate and mix by repeated aspiration        and dispensing of the solution into the wells.    -   7. Centrifuge the plate at 2000 RPM for 5 minutes to remove air        pockets in the wells.    -   8. Read in Tecan Polarion Plate Reader at 485 nm to obtain the        anisotropy value.

Target Protein 20 nM Peptide HDB Row Solution (μL) Solution (μL)Solution(μL) A 0 8 8 B 0 8 0 C 16 μL Row D 8 0 D 16 μL Row E 8 0 E 16 μLRow F 8 0 F 16 μL Row G 8 0 G 16 μL Row H 8 0 H 16 μL Row I 8 0 I 16 μLRow J 8 0 J 16 μL Row K 8 0 K 16 μL Row L 8 0 L 16 μL Row M 8 0 M 16 μLRow N 8 0 N 16 μL Row O 8 0 O 16 μL Row P 8 0 P 12 μL Target 0 ProteinSolution + 12 μL 40 nM Peptide Solution 0

The anisotropy (in mP—millipolarization units) vs the logarithm of theconcentration (micromoles/liter) of the receptor concentration isgraphed. The dissociation constant is obtained at the midpoint of thecurve whose extrema are A_(free) and A_(bound) where A_(free) is theanisotropy of the free peptide and A_(bound) is the anisotropy of thefully bound peptide. The theory, methods of operation and mathematicalanalysis is described in, for example, the following reference:Fluorescence Polarization Technical Resource Guide Technical ResourceGuide 4^(th) Edn. Invitrogen Corporation•501 Charmany Drive•Madison,Wis. 53719 USA. Particularly, the mathematical analysis of the data andobtaining binding constants is described in Chapter 8: Analysis of FPBinding Data pp 8-2-8-7.

Assay of Fibrin Binding Peptides by Competition Binding FluorescencePolarization (FP) Procedure using Competition of Unlabeled Test Peptidesvs the Standard 5-Carboxyfluoresein Labeled Peptide Seq000-CF5.

DDE at 10⁻⁵M concentration was aliquoted into a 96-well plate. Thestandard 5CF-labeled peptide Seq000-CF5 was added to DDE-containingwells to provide an initial tracer concentration of 10⁻⁶M. An aliquot ofcompetitor peptide was added to each well in order to span aconcentration range of 10⁻¹⁰M to 10⁻³M. The peptides were incubated withthe DDE/Seq000-CF5 complex for 2 h. Then the anisotropy value was readon the Tecan Polarion Plate Reader at 485 nm. The competition curve wasconstructed using all of the concentrations of the test peptide. Themathematical analysis of the data and calculation of the IC₅₀ for thetest peptides was accomplished using the regression routines in PrismGraph Pad™ Software. The theoretical and mathematical basis for theexperimental procedure and the data analysis is given in, for example:“Practical Use of Fluorescence Polarization In Competitive ReceptorBinding Assays”-Section P of “Receptor Binding Assays”http://www.Ncgc.Nih.Gov/Guidance/Section5.Html#Practical-Fluor-Polar.Copyright© 2005, Eli Lilly and Company and the National Institutes OfHealth Chemical Genomics Center. The relative IC50 values for testpeptides vs Seq000-CF5 were obtained by division of their IC50 by thatobtained by titration of Seq005 into Seq005-CF5/DDE complex. Thus lowerrelative IC50 indicates a stronger binding peptide. See Tables 1 and 2,supra.

Examples 1-3 below describe the preparation of exemplary peptidesSeq016, Seq017, and Seq049.

Example 1 Preparation of Ac-GWQPC*PWESWTFC*WDPGGGK-NH₂ cyclic (5→13)peptide (Seq016) (SEQ ID NO. 7)

The peptide sequence was prepared by SPSS from Fmoc-PAL-PEG-PS resin(0.18 mmol/g, 1.38 g, 0.25 mmol) as described in procedure A using anABI peptide synthesizer employing Fmoc chemistry which was implementedusing the FastMoc™ protocol. Cleavage and side-chain deprotection wasconducted as described in procedure G and disulfide cyclization wasaccomplished as described in procedure H. HPLC purification provided 105mg (17.8% yield) of the purified cyclic peptide.

Example 2 Preparation of Ac-SGSGJWQPC*PWESWTFC*WDPGGGK-NH₂ (cyclic 9→17)peptide (Seq017) (SEQ ID NO. 9)

Procedures A, G and H were employed to prepare the peptide on a 0.266mmol scale and HPLC purification provided 130 mg (18.8% yield) of thepure product.

Example 3 Preparation of Ac-RWQPC*PAESWT-Cha-C*WDPGGGK-NH₂ cyclic (5→13)peptide (Seq049) (SEQ ID NO. 69)

The peptide was prepared using the methods of procedures A, G and H.HPLC purification provided a 140 mg (27.5% yield) portion of the productas a fluffy white solid.

Example 4 below and FIG. 1 describe and illustrate the process used forthe preparation of Adoa-Adoa linker functionalized Seq005.

Example 4 Preparation of Ac-WQPC*PAESWTFC*WDPGGGK(JJ)-NH₂ cyclic (4→12)peptide, (Seq005-JJ) (SEQ ID NO. 136)

The ivDde-protected peptideAc-W(N^(in)-Boc)-Q(Trt)-P-C(Trt)-P-A-E(OtBu)-Ser(tBu)-W(N^(in)-Boc)-T(tBu)-F-C(Trt)-W(N^(in)-Boc)-D(OtBu)-P-GGGK(ivDde)-NH-TGRwas assembled on a 130 μmol scale (0.65 g resin) (procedure A). TheivDde group was removed (procedure D) by treatment of the resin with 10%hydrazine in DMF (6.5 mL) for 10 min (2×). Then the resin was washedwith DMF (4×). In a separate flask Fmoc-Adoa (100 mg, 0.26 mmol, 2.0equiv) in NMP (1 mL) was treated with HATU (99 mg, 0.26 mmol, 2 equiv)in DMF (0.5 mL) and DIEA (67 mg, 91 μL, 0.52 mmol, 4 equiv) for 2 minafter which the solution was transferred to the vessel containing theresin followed by agitation of the vessel for 12 h at ambienttemperature (procedure F).

The resin was washed with DMF (4×5 mL) and the Fmoc group was removed bytreatment with 20% piperidine in DMF (10 mL, 2×10 min) followed bywashing (4×10 mL) with DMF (procedure C). Then Fmoc-Adoa was coupled tothe resin as described (vide supra) followed by removal of the Fmocprotecting group (vide supra) and washing of the resin. Cleavage andside-chain deprotection (procedure G) was conducted for 4.5 h usingReagent B (10 mL). The resin was drained and washed with TFA (5 mL) andthe combined solutions were evaporated and triturated with ether toprovide the crude linear peptide as an off-white solid. The solid wasdissolved in DMSO (3 mL) after which the pH of the solution was adjustedto 8 by addition of 0.1M aqueous N-methylglucamine. The mixture wasstirred for 48 h (procedure H) during which time the reaction wasmonitored by analytical HPLC and mass spectroscopy. At the end of thereaction period the entire solution was diluted to 15 mL with 10% CH₃CNin H₂O and the pH was adjusted to 2 by addition of aqueous TFA.

The resulting solution was applied to a preparative reverse-phase C18column and purified using a linear gradient of 10% CH₃CN (0.1% TFA) intoH₂O (0.1% TFA). Fractions (15 mL) were collected and the pureproduct-containing fractions were pooled, frozen and lyophilized toprovide 42 mg (13% yield) of the peptide as a fluffy white solid whichwas characterized by HPLC and mass spectroscopy. HPLC: t_(R) 3.83 min;Column: Waters XTerra MS-C18 4.6×50 mm; Particle size: 5 microns;Eluents: A: Water (0.1% TFA), B: Acetonitrile (0.1% TFA); Elution:linear gradient 5-65% B in 7 min; Flow rate: 3 mL/min; Detection: UV,λ=220 nm. Mass spectrum (API-ES): Neg. ion: [M-H]: 2480.6; [M-2H]/2:1239.9

Examples 5 and 6 below describe the preparation of peptides bearingN-terminal Aloc-Arginine.

Example 5 Preparation of Aloc-RWQPC*PWESWTFC*WDPGGGK-NH₂ cyclic (5→13)peptide (Seq023-Aloc) (SEQ ID NO. 22)

A 0.54 mmol (3 g) portion ofW(N^(in)-Boc)-Q(Trt)-P-C(Trt)-P-W(N^(in)-Boc)-E(OtBu)-S(tBu)-W(N^(in)-Boc)-T(tBu)-F-C(Trt)-W(N^(in)-Boc)-D(OtBu)-P-GGG-K(Boc)-PAL-PEG-PSresin was prepared by automated SPSS (procedure A). Aloc-Arg(Pmc) wasappended to the N-terminus using a modification of procedure B asfollows: The resin was added to a manual solid phase synthesis vesseland suspended in DMF (20 mL) by brief agitation. Aloc-Arg(Pmc)-OH (524mg, 1.00 mmol, 1.85 equiv), HATU (380 mg, 1.0 mmol, 1.85 equiv) and DIEA(257 mg, 347 μL, 1.98 mmol, 3.67 equiv) were added successively withintervening agitation of the vessel and the vessel was shaken overnight.The coupling reaction was complete as indicated by a negative ninhydrintest. The resin was washed with DCM (3×20 mL) and dried.

Reagent B (88:5:5:2-TFA: water: phenol: TIPS-v/v/wt/v) (25 mL) was addedto the vessel and the vessel was shaken at ambient temperature for 5 h.The resin was filtered and washed with TFA (2×5 mL). The combinedfiltrates were concentrated to a syrup which was triturated with Et₂O(20 mL) and the resulting solid was pelleted by centrifugation. Thesupernatant liquid was decanted and the process was repeated three times(procedure G). The resulting solid was collected and cyclized (48 h) asdescribed (procedure H) and purified by HPLC on a reverse phase C18column. The product-containing fractions were pooled, frozen andlyophilized to provide 290 mg (21% yield) of the desired product.

Example 6 Preparation of Aloc-RWQPC*PAESWT-Cha-C*WDPGGGK-NH₂(Seq057-Aloe) (SEQ ID NO. 86)

The peptide was prepared by the methods of procedure A, B, G and H togive 230 mg (26.7% yield) portion of the product as a fluffy whitesolid.

Example 7 below and FIG. 2 describe and illustrate the preparation of5-carboxyfluorescein derivatives of peptides.

Example 7 Preparation of RWQPC*PAESWTFC*WDPGGGK(CF5)-NH₂ cyclic (5→13)peptide (Seq056-CF5) (SEQ ID NO. 120)

The peptide Aloc-RWQPC*PAESWTFC*WDPGGGK-NH₂ cyclic (5→13) peptide(Seq056-Aloc) (SEQ ID NO. 140) was prepared by the methods of proceduresA, B, G and H and purified by HPLC. The N-terminal Aloc N^(ε20)-CF5derivative was prepared according to procedure I as follows: The peptide(70 mg, 0.029 mmol) was dissolved in anhydrous DMF (1 mL) with stirring,after which DIEA (0.074 g, 100 μL, 0.572 mmol, 19.7 equiv) was addedfollowed by a solution of CF5-NHS (20 mg, 0.042 mmol, 1.45 equiv) inanhydrous DMF. The mixture was stirred 1 h at ambient temperature. Thereaction mixture was diluted to twice its volume with 20% CH₃CN in H₂Oand purified on a C18 reverse phase preparative HPLC column to provide50 mg (62.8% yield) of Aloe-RWQPC*PAESWTFC*WDPGGGK(CF5)-NH₂ (SEQ ID NO.141) cyclic (5→13) peptide.

The Aloc group of this intermediate was removed according to procedure Las follows: The intermediate was dissolved in a solution of NMM:HOAc:DMF(1:2:10, v/v/v, 5 mL), the mixture was stirred and Pd(PPh₃)₄ (21 mg,0.018 mmol, 1.0 equiv) was added. The mixture was stirred 1 h at ambienttemperature. Then the reaction mixture was diluted to twice its volumewith 10% CH₃CN in H₂O and purified on a preparative reverse phase C18column using a linear gradient of CH₃CN (0.1% TFA) into H₂O (0.1% TFA).The pure product-containing fractions were pooled, frozen andlyophilized to provide 29 mg (60.4% yield) of the product as an orangesolid.

Examples 8-16 below and FIGS. 3-5 describe and illustrate thepreparation of lipopeptides, particularly DSPE-PG4-peptide conjugates,DPPE-PG4-peptide conjugates, DPPE-PG2-peptide conjugates, andDPPE-Pro9-Glut-Ttda-Dga-peptide conjugates. The following Table 5 setsforth the MS and analytical data for the lipopeptides:

TABLE 5 HPLC Data MALDI Mass Spectral (System, Data Sequence t_(R))(Mode: Ions) Seq005- Ac-WQPC*PAESWTFC*WDPGGGK(DSPE-PG4-Glut)-NH₂I,  5.612 Pos. Ion: [M + H]: 5124 PL1 (SEQ ID NO. 123) Seq005-Ac-WQPC*PAESWTFC*WDPGGGK(DPPE-PG4-Glut-)-NH₂ I,  5.361 Pos. Ion: [M +H]: 5026 PL2 (SEQ ID NO. 124) Seq024-Ac-SGSGSGSGWQPC*PWESWTFC*WDPGGGK(DSPE- J,  9.25 Pos. Ion: 5753 [M + H],PL1 PG4-Glut)-NH₂ 1918 [M + 3H]/3, 1439 (SEQ ID NO. 125) [M +4H]/4, 1178 [M + 5H]/5 Seq016- Ac-GWQPC*PWESWTFC*WDPGGGK(DSPE-PG4-Glut)-K, 11.40 Pos. Ion: [M + H]: 5233 PL1 NH₂ (SEQ ID NO. 126) Seq017-Ac-SGSGJWQPC*PWESWTFC*WDPGGGK(DSPE-PG4- K, 10.67 Pos. Ion: [M + H]: 5565PL1 Glut)-NH₂ (SEQ ID NO. 127) Seq023-RWQPC*PWESWTFC*WDPGGGK(DSPE-PG4-Glut)-NH₂ L, 15.42 Pos. Ion: [M +H]: 5290 PL1 (SEQ ID NO. 128) Seq049-Ac-RWQPC*PAESWT-Cha-C*WDPGGGK(DSPE-PG4- M,  7.12 Pos. Ion: [M + H]:  522PL1 Glut)-NH₂ (SEQ ID NO. 129) Seq057-RWQPC*PAESWT-Cha-C*WDPGGGK(DSPE-PG4-Glut)- N, 14.05 Pos. Ion: [M +H]: 5180 PL1 NH₂ (SEQ ID NO. 130) Seq005-Ac-WQPC*PAESWTFC*WDPGGGK(DPPE-Glut-PG2-JJ)- O,  5.97 Pos. Ion: [M +H]: 6656 PL3 NH₂ (SEQ ID NO. 131) Seq005-Ac-WQPCPAESWTFCWDPGSAGSK(DPPE-Pro9-Glut- P,  5.68 Neg. ion: [2M − 3H]/3:PL4 Ttda-Dga)-NH₂ 2905.5, [M + Na − 3H]/2: (SEQ ID NO. 132) 2189.6, [M −2H]/2: 2178.4, [M − 3H]/3: 1452.2 Seq005-Ac-WQPCPAESWTFCWDPGAGSGK(DPPE-Pro9-Glut- P,  5.77 Neg. ion: [2M − 3H]/3:PL5 Ttda-Dga)-NH₂ 2884.2, [M − 2H]/2: 2163.3, (SEQ ID NO. 133) [M −3H]/3: 1441.3

Example 8 Preparation of Ac-WQPC*PAESWTFC*WDPGGGK(DSPE-PG4-Glut)-NH₂cyclic (4→12) peptide (Seq005-PL1) (SEQ ID NO. 123)

A solution of the peptide Ac-WQPC*PAESWTFC*WDPGGGK-NH₂ cyclic (4→12)peptide (Seq005) (SEQ ID NO.1) (150 mg, 0.069 mmol) in DMF (1.0 mL) wasadded to a stirred solution of DSG (0.34 mmol, 112 mg, 5 equiv) and DIEA(15 mg, 20 μL, 0.12 mmol, 1.67 equiv) in DMF (1.0 mL). The mixture wasstirred for 0.5 h and the progress of the reaction was monitored by HPLCand MS. Upon completion of the reaction, the volatiles were removed invacuo and the residue was washed with ethyl acetate (3×10 mL) to removeunreacted DSG. The residue was dried, re-dissolved in anhydrous DMF (1.0mL) and a solution of DSPE-PG4-NH₂ (134 mg, 0.048 mmol) in DMF (1.0 mL)was added followed by DIEA (15 mg, 20 μL, 0.12 mmol, 1.67 equiv). Themixture was stirred for 16 h. The progress of the reaction was monitoredby HPLC which indicated that the aminopegylated phospholipid wasconsumed at 16 h.

The reaction mixture was diluted with distilled, deionized water andpurified on a reverse phase preparative column (YMC Prep C₄, particlesize 10 μm, 30×250 mm), using a gradient of 50-75% water (0.1% TFA) intoCH₃OH:CH₃CN (1:1, v/v, 0.1% TFA) at a flow rate of 30 mL/min over aperiod of 5 min then ramping to 100% B over 50 min. Fractions (15 mL)were analyzed by HPLC and the pure product-containing fractions werepooled. Methanol was removed from the combined product-containingeluates by rotary evaporation; the resulting solution was diluted with10% aqueous acetonitrile, frozen and lyophilized to provide 108 mg (44%yield) of the desired product as a fluffy white solid.

Example 9 Preparation ofAc-SGSGSGSGWQPC*PWESWTFC*WDPGGGK(DSPE-PG4-Glut)-NH₂ cyclic (12→20)peptide (Seq024-PL1) (SEQ ID NO. 125)

A solution of DSG (50 mg, 0.15 mmol, 4.41 equiv) and DIEA (20 mg, 0.15mmol, 4.41 equiv) in anhydrous DMF (2.0 mL) was stirred and the peptideAc- SGSGSGSGWQPC*PWESWTFC*WDPGGGK-NH₂ cyclic (1220) peptide (Seq024)(SEQ ID NO. 23) (100 mg, 0.034 mmol) was added in solid form portionwiseto the above solution. The mixture was stirred at room temperature for30 min. The volume of the reaction mixture was adjusted to 50 mL byaddition of EtOAc and the precipitated solid was pelleted bycentrifugation followed by decantation of the supernatant. The washingprocedure was repeated (3×) to provide the glutaric acid monoamidemono-NHS ester of the peptide, whose identity was confirmed by massspectral analysis [(M-2H)/2: 1546.1, (M-3H)/3: 1030.5], as a colorlesssolid.

The glutaric acid monoamide mono-NHS ester of the peptide was dissolvedin dry DMF-DCM (2.0 mL, 8:2, v/v). DIEA (40 mg, 54 μL, 0.31 mmol) wasadded and the mixture was stirred. DSPE-PG4-NH₂ (45 mg, 0.038 mmol, 0.9equiv) was added as a solid and the mixture was stirred for 24 h atambient temperature. The volume of the mixture was adjusted to 100 mL byaddition of CH₃OH (50%) and CH₃CN—water (1:1) (50%) and the resultingsolution was filtered to remove insoluble material.

The filtered solution was loaded onto a C4 reverse phase column (YMC,Prep C₄, 10 μM, 100 Å, 30×250 mm) which had been pre-equilibrated with50% CH₃OH and CH₃CN (eluent A)—water (eluent B) (1:1) at 30 mL/min. Thecolumn was washed with the same eluent until the plug of DMF was elutedfrom the column. The mobile phase composition was then ramped to 70% Bin 1 min and the elution was continued at a linear gradient rate of 1%B/min to 100% B at which time the column was eluted with 100% B untilthe product was fully eluted from the column. Fractions (15 mL) werecollected and those containing the product in >98% purity were collectedand concentrated on a rotary evaporator to reduce the content of CH₃OH.The concentrated solution was diluted with 10% CH₃CN in water, frozenand lyophilized to provide 65 mg (60% yield) of the product as acolorless solid.

Example 10 Preparation of Ac-GWQPC*PWESWTFC*WDPGGGK(DSPE-PG4-Glut)-NH₂cyclic (5→13) peptide (Seq016-PL1) (SEQ ID NO. 126)

The peptide Ac-GWQPC*PWESWTFC*WDPGGGK-NH₂ cyclic (5→13) peptide (Seq016)(SEQ ID NO. 7) (90 mg, 0.038 mmol) was dissolved in anhydrous DMF (0.5mL) and this solution was added to a solution of DSG (65 mg, 0.2 mmol,5.26 equiv) and DIEA (25 mg, 33.8 μL, 0.2 mmol, 5.26 equiv) in anhydrousDMF (0.5 mL) with stirring. The mixture was stirred 2 h and then EtOAc(20 mL) was added resulting in the formation of a solid which waspelleted by centrifugation. The supernatant liquid was decanted and thisprocess was repeated twice to remove remaining DSG. The solid was driedunder vacuum (<0.1 mm) for 30 min followed by dissolution by stirring inDMF (0.5 mL). Solid DSPE-PG4-NH₂ (53 mg, 0.19 mmol) was addedportionwise and the resulting mixture was stirred overnight. The mixturewas diluted with water (5 mL) and the crude mixture was purified bypreparative HPLC to give 60 mg (30% yield) of the desired product as awhite lyophilizate.

Example 11 Preparation ofAc-SGSGJWQPC*PWESWTFC*WDPGGGK(DSPE-PG4-Glut)-NH₂ cyclic (9→17) peptide(Seq017-PL1) (SEQ ID NO. 127)

The peptide Ac-SGSGJWQPC*PWESWTFC*WDPGGGK-NH₂ cyclic (9→17) peptide(Seq017) (SEQ ID NO. 9) (100 mg, 0.039 mmol) was employed using theprocedures described for the preparation of SEQ005-PL1. HPLCpurification provided 53 mg (25.7% yield) of the targetphospholipid-peptide conjugate.

Example 12 Preparation of RWQPC*PWESWTFC*WDPGGGK(DSPE-PG4-Glut)-NH₂cyclic (5→13) peptide (Seq023-PL1) (SEQ ID NO. 128)

Aloc-RWQPC*PWESWTFC*WDPGGGK-NH₂ cyclic (5→13) peptide (Seq023-Aloc) (SEQID NO. 142) (100 mg, 0.04 mmol) in DMF (0.5 mL) was added to a solutionof DSG (50 mg, 0.153 mmol, 3.825 equiv) in DMF (0.5 mL). DIEA (7.4 mg,10 μL, 0.057 mmol, 1.43 equiv) was added to the solution and stirringwas continued for 1 h at ambient temperature after which massspectroscopy indicated completion of the reaction. The volatiles wereremoved under high vacuum to provide a semisolid residue. EtOAc (5 mL)was added to the residue resulting in the formation of a well-definedsolid which was pelleted by centrifugation. The supernatant liquid wasdecanted and the washing process was repeated (5×). This gave a 100 mg(92% yield) portion of a white solid, the intermediate N^(ε19)-glutaricacid monoamide mono-NHS ester of the peptide. A second run, conducted inexactly the same manner, provided an additional 100 mg of white solid.Mass spectral analysis was consistent with the presumed structure of theintermediate. The calculated monoisotopic molecular weight of theintermediate NHS ester is 2713. Mass spectral analysis (API-ES negativeion) of the white solid obtained by the above described procedure gavepeaks at 1356 [(M-2H)/2] and 1431.6 [(M+TFA-2H)/2].

The glutaric acid monoamide, mono-NHS ester of the peptide (200 mg,0.074 mmol) was dissolved in DMF (1 mL), and DIEA (11 mg, 15 μL, 0.085mmol, 1.15 equiv) was added to the stirred solution. DSPE-PG4-NH₂ (160mg, 0.8 equiv) in DMF (1 mL) was added to the mixture and stirring wascontinued overnight at ambient temperature. The volatiles were removedat high vacuum. The residue was re-dissolved in 13 mL of NMM/HOAc/DMF(1:2:10, v/v/v). Pd(PPh₃)₄ (300 mg, 3.0 equiv) was added in one portion.The mixture was stirred for 1 h. MS and analytical HPLC indicatedcompletion of the reaction. The crude reaction mixture was diluted withan equal volume of 20% CH₃CN in H₂O and insoluble material was filtered.The resulting solution was applied directly to a C4 reverse phase HPLCcolumn and purified by preparative HPLC as described for Seq005-PL1.Fractions containing the pure product were pooled, frozen andlyophilized to give 140 mg (29% yield based on the input DSPE-PG4-NH₂)of the target compound as a fluffy solid.

Example 13 Preparation ofAc-RWQPC*PAESWT-Cha-C*WDPGGGK(DSPE-PG4-Glut)-NH₂, cyclic (5→13) peptide(Seq049-PL1) (SEQ ID NO. 129)

Ac-RWQPC*PAESWT-Cha-C*WDPGGGK-NH₂ (SEQ ID NO. 70) cyclic (5→13) peptide(94 mg, 0.04 mmol, 1.25 equiv) was employed to prepare a 57 mg (34%yield) portion of the target phospholipid peptide conjugate in the samemanner as Seq005-PL1.

Example 14 Preparation of RWQPC*PAESWT-Cha-C*WDPGGGK(DSPE-PG4-Glut)-NH₂cyclic (5→13) peptide (Seq057-PL1) (SEQ ID NO. 130)

A solution of Aloc-RWQPC*PAESWT-Cha-C*WDPGGGK-NH₂ cyclic (5→13) peptide(Seq057-Aloc) (SEQ ID NO. 143) (230 mg, 0.096 mmol) in anhydrous DMF (1mL) was added dropwise to a stirred solution of DSG (180 mg, 0.55 mmol,5.75 equiv relative to peptide) in anhydrous DMF (0.5 mL) containingDIEA (110 mg, 0.86 mmol, 8.9 equiv relative to peptide). The reactionmixture was stirred for 3 h and the progress of the reaction wasmonitored by analytical HPLC and mass spectroscopy. The solvents wereremoved under high vacuum at 30° C. to give a viscous residue. Theresidue was triturated by addition of EtOAc (15 mL) to the vessel,resulting in the formation of a white solid which was pelleted bycentrifugation. The supernatant was decanted and the solid washed (3×)in the same manner and dried using a flow of dry nitrogen gas.

The solid intermediate thus obtained was dissolved in anhydrous DMF (1mL) with stirring and DIEA (110 mg, 149 μL, 0.86 mmol, 8.9 equivrelative to peptide) was added, followed by the dropwise addition of asolution of DSPE-PG4-NH₂ (213 mg, 0.077 mmol, 0.8 equiv) in anhydrousDMF (1 mL). The mixture was stirred at ambient temperature overnight,after which HPLC analysis and mass spectroscopy indicated theconsumption of the lipid.

The volatiles were removed under high vacuum and the crude mixture wasdissolved in a solution of 13 mL of NMM:HOAc:DMF (1:2:10, v/v/v) withstirring. Pd(PPh₃)₄ (250 mg, 0.22 mmol, 2.25 equiv relative to peptide)was added to the stirred solution and stirring was continued for 4 h.The solids were filtered and the resulting solution was diluted to twiceits volume with a solution of 25% CH₃CN in H₂O. The resulting solutionwas loaded onto a Zorbax C-3 column (21.2 mm i.d.×150 mm) which waspre-equilibrated with 25% B (CH₃CN with 0.1% TFA) at a flow rate of 30mL/min. The column was eluted at 30 mL/min with the same eluent untilthe plug of DMF was eluted. The proportion of eluent B was thenincreased from 25% B to 30% B over 3 min and then ramped to 100% B over50 min. Fractions (15 mL) were collected and product-containingfractions were pooled, frozen and lyophilized. HPLC analysis of theisolated product indicated the need for additional purification. Thepurification was repeated and the product was isolated by freezing thepooled pure product-containing fractions and lyophilization to provide23 mg (4.5% yield) of the target phospholipid-peptide conjugate.

Example 15 below describes the preparation of lipopeptideDPPE-PG4-peptide conjugates.

Example 15A Preparation of Ac-WQPC*PAESWTFC*WDPGGGK(DPPE-PG4-Glut)-NH₂cyclic (4→12) peptide (Seq005-PL2) (SEQ ID NO. 124)

The process is adapted from that used for the preparation of Seq005-PL1except that DPPE-PG4-NH₂ is employed in place of DSPE-PG4-NH₂. Thus asolution of the peptide Ac-WQPC*PAESWTFC*WDPGGGK-NH₂ cyclic (4→12)peptide (Seq005) (SEQ ID NO. 1) (66 mg, 0.03 mmol) and DIEA (11 mg, 15μL, 0.085 mmol, 2.84 equiv) in DMF (0.5 mL) was added to a stirredsolution of DSG (33 mg, 0.10 mmol, 3.3 equiv) in DMF (0.5 mL). Themixture was stirred for 1 h and the progress of the reaction wasmonitored by HPLC and MS. Upon completion of the reaction, the volatileswere removed in vacuo and the residue was washed with ethyl acetate (4×5mL) to remove unreacted DSG from the intermediate peptide glutaric acidmonoamide mono-NHS ester.

The resulting solid was dried, re-dissolved in anhydrous DMF (1.00 mL)and stirred with a solution of DPPE-PG4-NH₂ (95 mg, 0.035 mmol, 1.16equiv) in DMF (1.0 mL) for 24 hour. The progress of the reaction wasmonitored by HPLC, which indicated complete consumption of theintermediate peptide glutaric acid monoamide mono-NHS ester. Thesolution was diluted with water and purified by reverse phase C4preparative column (Kromasil® Prep C₄, 10 μm, 300 Å, 20×250 mm, flowrate 25 mL/min) using a gradient of 60-100% water (0.1% TFA) and amixture of CH₃OH and CH₃CN (1:1, 0.1% TFA) over a period of 30 min.Fractions were collected in 15 mL portions and analyzed by HPLC (Column:YMC C-4, 5 μm, 300 Å, 4.6×250 mm) using an ELSD and a UV detector (λ=220nm). Pure product-containing fractions were collected and concentratedon a rotary evaporator to remove the methanol from the eluate and theresulting solution was diluted with 10% CH₃CN in H₂O, frozen andlyophilized to afford 72 mg (48% yield) of the required product as afluffy white solid.

Example 15B Preparation of Comparative Peptide Seq000 conjugated toDSPE-PG4-Glut, referred to as Seq000-PL1

Ac-WQPC*PWESWTFC*WDPGGGK(DPPE-PG4-Glut)-NH₂ cyclic (4→12) peptideSeq000-PL1 was prepared in a similar manner to the procedure of Example15A. Thus a solution of the peptide Ac-WQPC*PWESWTFC*WDPGGGK-NH₂ cyclic(4→12) peptide (Seq000) (SEQ ID NO. 122) (115 mg, 0.05 mmol) and DIEA(130 mg, 176 μL, 1.0 mmol, 20 equiv) in DMF (2.0 mL) was added to astirred solution of DSG (100 mg, 0.30 mmol, 6 equiv) in DMF (0.5 mL).The mixture was stirred for 1 h and the progress of the reaction wasmonitored by HPLC and MS. Upon completion of the reaction, the volatileswere removed in vacuo and the residue was washed with ethyl acetate(4×20 mL) to remove unreacted DSG from the intermediate peptide glutaricacid monoamide mono-NHS ester.

The resulting solid was dried, re-dissolved in anhydrous DMF (1.0 mL)and stirred with a solution of DPPE-PG4-NH₂ (160 mg, 0.0573 mmol, 1.11equiv) in DMF (1.0 mL) for 24 hour. The progress of the reaction wasmonitored by HPLC, which indicated complete consumption of theintermediate peptide glutaric acid monoamide mono-NHS ester. Thesolution was diluted with water and purified by reverse phase C4preparative column (Kromasil® Prep C₄, 10 μm, 300 Å, 20×250 mm, flowrate 25 mL/min) using a gradient of 50-100% water (0.1% TFA) and amixture of CH₃OH and CH₃CN (1:1, 0.1% TFA) over a period of 30 min.Fractions were collected in 15 mL portions and analyzed by HPLC (Column:YMC C-4, 5 μm, 300 Å, 4.6×250 mm) using an ELSD and a UV detector (λ=220nm). Pure product-containing fractions were collected and concentratedon a rotary evaporator to remove the methanol from the eluate and theresulting solution was diluted with 10% CH₃CN in H₂O, frozen andlyophilized to afford 138 mg (52% yield) of the required product as afluffy white solid.

Mass Spectrum: Method: MALDI, Mode: Positive Ion, [M+Na+2H]: 5199(Average)

HPLC: Ret. time: 5.88 min; Assay: >98% (area %); Column: YMC C-4, 5 μM,300 Å, 4.6×50 mm; Eluents: A: Water (0.1% TFA), B: Acetonitrile:Methanol (1:1, v/v)(0.1% TFA); Elution: Initial condition: 80% B, lineargradient 80-100% B in 10 min.; Flow rate: 3.0 mL/min; Detection: UV @220 nm & ELSD.

Example 16A below and FIG. 5 describe and illustrate the preparation ofDPPE-Glut-PG2-peptide conjugates.

Example 16A Preparation ofAc-WQPC*PAESWTFC*WDPGGGK(DPPE-Glut-PG2-JJ)-NH₂ cyclic (4→12) peptide(Seq005-PL3) (SEQ ID NO. 131)

The peptide, Ac-WQPC*PAESWTFC*WDPGGGK(JJ)-NH₂ cyclic (4→12) peptide(Seq005-JJ) (SEQ ID NO. 136) (63 mg, 0.025 mmol) was dissolved in DMF(1.0 mL) and DIEA (9.8 mg, 13.3 μL, 0.076 mmol, 3 equiv) was added. Thesolution was stirred briefly and a solution of Fmoc-PG2-NHS (111 mg,0.032 mmol, 1.28 equiv) in DMF (1.0 mL) was added dropwise withstirring. The mixture was stirred for 16 h and HPLC analysis indicatedthe consumption of the starting peptide. The reaction mixture wastreated with piperidine (172 mg, 200 4, 2.02 mmol, 80 equiv, finalconcentration ˜9% v/v in the reaction mixture) for 30 min after whichthe reaction mixture was diluted with H₂O and purified by preparativeHPLC employing a reverse phase C4 column (Kromasil® Prep C₄, 10 μm, 300Å, 20×250 mm) by the following method: elution: the plug of DMF waseluted at the initial condition of 20% CH₃CN in H₂O (0.1% TFA), then alinear gradient of 20%-80% CH₃CN (0.1% TFA) into H₂O (0.1% TFA) over aperiod of 40 min at a flow rate of 25 mL/min was employed to elute theproduct. Fractions (15 mL) were collected and those containing the pureproduct (HPLC analysis) were pooled, frozen and lyophilized to provide70 mg (47% yield) of the PG2-derivatized peptideAc-WQPC*PAESWTFC*WDPGGGK(NH₂-PG2-JJ) (SEQ ID NO. 143) —NH₂ cyclic (4→12)peptide.

A solution of Ac-WQPC*PAESWTFC*WDPGGGK(NH₂-PG2-JJ)-NH₂ (SEQ ID NO. 143)cyclic (4→12) peptide (50 mg, 0.009 mmol, 1.14 equiv) in DMF (1.0 mL)was treated with DIEA (14.8 mg, 20 μL, 0.114 mmol, 14.6 equiv) followedby DPPE-Glut-NHS (7 mg, 0.0078 mmol) in a 1:1 mixture of DMF:CH₂Cl₂ (1.0mL) and the mixture was stirred for 16 hr at room temperature. Thesolution was diluted with H₂O to 10 mL and purified by reverse phase C4preparative column (Kromasil® Prep C₄, 10 μm, 300 Å, 10×250 mm, flowrate, 10 mL/min) using a gradient of 50-80% water (0.1%TFA)/acetonitrile:MeOH (1:1, 0.1% TFA) over a period of 40 min. The pureproduct-containing fractions were pooled, frozen and lyophilized toafford 26 mg (42% yield) of the target compound as a fluffy white solid.

Examples 16B-16C below describe and illustrate the preparation ofDPPE-Pro9-Glut-Ttda-Dga-peptide conjugates. Example 16D below describesand illustrates the preparation of a comparative lipopeptide,Seq000-PL2, from Seq000(Adoa-Adoa) and DPPE.

Example 16B Preparation ofAc-WQPCPAESWTFCWDPGSAGSK(DPPE-Pro9-Glut-Ttda-Dga)-NH₂ cyclic (4-12)peptide (Seq005-PL4) (SEQ ID NO. 132)

Preparation of Seq005-P2(Ttda-Dga)

Chain elongation of the peptide was conducted on 1.2 g ofFmoc-Pal-Peg-PS resin (0.17 mmol/g) on a 0.2 mmol scale (procedure A) toprovide Ac-W(N^(in)-Boc)-Q(Trt)-P-C(Trt)-P-A-E(OtBu)-S(tBu)-W(N^(in)-Boc)-T(tBu)-F-C(Trt)-W(N^(in)-Boc)-D(OtBu)-P-GS(tBu)-A-G-S(tBu)-K(ivDde)-NH-Pal-Peg-PS resin. Resins from two runs were combined. TheivDde group was removed using 20 mL of 10% hydrazine in DMF (2×10 min).Then diglycolic anhydride (0.464 g, 10 eq) and DIEA (0.516 g, 0.7 mL,4.0 mmol) in DMF (20 mL) were added to the resin and the resin wasagitated for 15 h. The resin was washed with DMF (5×20 mL) and thentreated with N1-(tert-butoxycarbonyl)-1,3-diamino-4,7,10-trioxatridecane(0.513 g, 1.6 mmol, 4 eq), HATU (0.608 g, 1.6 mmol, 4 eq) and DIEA(0.413 g, 0.558 mL, 3.2 mmol, 8 eq) in DMF (20 mL) for 15 h. The resinwas washed with DMF (5×20 mL). The peptide was cleaved from the resinusing reagent B (procedure G) and the crude solid peptide was subjectedto disulfide cyclization (procedure H). The crude mixture was dilutedwith water to about five-fold its volume and applied to a Waters XTerraC-18 (250 mm×50 mm i.d.) column and purified using a linear gradientelution of ACN (0.1% TFA) into H₂O (0.1% TFA) as described in theprocedure titled. The pure product-containing fractions were pooled,frozen and lyophilized to provide 175 mg (16.2% yield) of the peptide asa fluffy white solid.

Preparation of Ac- WQPCPAESWTFCWDPGSAGSK(DPPE-Pro9-Glut-Ttda-Dga)-NH₂cyclic (4→12) peptide (Seq005-PL4) from DPPE-Pro9-H andSeq005-P2(Ttda-Dga)

DPPE-(Pro)₉-H, 120 mg, 0.077 mmol was added to DSG (100 mg, 0.306 mmol,4.0 eq) in DMF (2 mL), followed by DIEA (0.059 g, 0.08 mL, 0.46 mmol,6.0 equiv); the mixture was stirred 4 h. The volatiles were removedunder high vacuum. The resulting crude residue was washed twice withEtOAc to remove DSG and remaining traces of DMF and DIEA. The crude wasredissolved in DMF (1 mL) and Seq005-P2(Ttda-Dga)[Ac-WQPCPAESWTFCWDPGSAGSK(Dga-Ttda)-NH₂] (SEQ ID NO. 145), (170 mg,0.063 mmol) in DMF (1 mL) was added, followed by DIEA (0.088 g, 0.12 mL,0.69 mmol, 11 equiv relative to the added peptide). The mixture wasstirred at 40° C. for 15 h. The resulting mixture was diluted with 35%MeOH in water (25 mL) and filtered using a 0.45 micron filter. Thesolution was purified by preparative HPLC. The pure product containingfractions were combined, frozen and lyophilized to provide the product(90 mg, 32.8% yield) as a fluffy solid.

Example 16C Preparation ofAc-WQPCPAESWTFCWDPGAGSGK(DPPE-Pro9-Glut-Ttda-Dga)-NH₂cyclic (4→12)peptide (Seq005-PL5) (SEQ ID NO. 133)

Preparation of Seq005-P3(Ttda-Dga)

Chain elongation of the peptide was conducted on 1.2 g ofFmoc-Pal-Peg-PS resin (0.17 mmol/g) on a 0.2 mmol scale (procedure A) toprovide Ac-W(N^(in)-Boc)-Q(Trt)-P-C(Trt)-P-A-E(OtBu)-S(tBu)-W(N^(in)-Boc)-T(tBu)-F-C(Trt)-W(N^(in)-Boc)-D(OtBu)-P-G-A-G-S(t-Bu)-G-K(ivDde)-NH-Pal-Peg-PSresin. Resins from two runs were combined. The ivDde group was removedusing 20 mL of 10% hydrazine in DMF (2×10 min). Then diglycolicanhydride (0.464 g, 10 eq) and DIEA (0.516 g, 0.7 mL, 4.0 mmol) in DMF(20 mL) were added to the resin and the resin was agitated for 15 h. Theresin was washed with DMF (5×20 mL) and then treated withN1-(tert-butoxycarbonyl)-1,3-diamino-4,7,10-trioxatridecane (0.385 g,1.2 mmol, 3 eq), HATU (0.456 g, 1.2 mmol, 3 eq) and DIEA (0.310 g, 0.419mL, 2.4 mmol, 6 eq) in DMF (15 mL) for 15 h. The resin was washed withDMF (5×20 mL). The peptide was cleaved from the resin using reagent B(procedure G) and the crude solid peptide was subjected to disulfidecyclization (procedure H). The crude mixture was diluted with water toabout five-fold its volume and applied to a Waters XTerra C-18 (250mm×50 mm i.d.) column and purified using a linear gradient elution ofACN (0.1% TFA) into H₂O (0.1% TFA) as described in the procedure titled.The pure product-containing fractions were pooled, frozen andlyophilized to provide 227.7 mg (21.3% yield) of the peptide as a fluffywhite solid.

Preparation ofAc-WQPCPAESWTFCWDPGAGSGK(DPPE-Pro9-Glut-Ttda-Dga)-NH₂cyclic (4→12)peptide (Seq005-PL5) from DPPE-Pro9-H and Seq005-P3(Ttda-Dga)

DSG (75 mg, 0.230 mmol, 3.59 eq) in DMF (0.75 mL) was stirred and tothis mixture was added DPPE-(Pro)₉-H (100 mg, 0.064 mmol) dissolved inDCM (0.5 mL). Then DIEA (0.03 g, 0.04 mL, 0.23 mmol, 3 equiv) was addedand the mixture was stirred 4 h. Mass spectral analysis confirmed theformation of the glutaric acid mono-amide-mono-NHS ester of DPPE-(Pro)₉.The volatiles were removed under high vacuum and the crude residue keptfor 2 h under high vacuum. The resulting crude residue was trituratedand washed with EtOAc to remove DSG and remaining traces of DMF andDIEA. The crude was redissolved in DCM (1 mL) and Seq005-P3(Ttda-Dga)[Ac-WQPCPAESWTFCWDPGSAGSK(Ttda-Dga)-NH₂] (SEQ ID NO. 145), (165 mg,0.062 mmol) in DMF (1 mL) was added, followed by DIEA (0.088 g, 0.12 mL,0.69 mmol, 11 equiv relative to the added peptide). The mixture wasstirred at 40° C. for 15 h after which HPLC and MS analysis indicatedformation of the desired product. The resulting mixture was diluted with35% MeOH in water (15 mL) and filtered using a 0.45 micron filter. Thesolution was purified by preparative HPLC on a C2 column. The compoundwas applied to the column at 25% ACN-MeOH 1:1, v/v (eluent B) in water(eluent A). After the compound was applied and the solvent plug eluted,the eluent composition was ramped to 50% B and then ramped from 50-100%B over 30 min. The pure product containing fractions were combined andmost of the MeOH was removed by rotary evaporation. Tert-Butyl alcoholwas added to the mixture and the mixture was frozen and lyophilized togive the product (125 mg, 46.5% yield) as a fluffy solid.

Example 16D Preparation of Comparative Lipopeptide Seq000-PL2

The fully side-chain protected peptide sequenceAc-W(Boc)-Q(Trt)-P-C(Trt)-P-WBoc)-E(O-t-Bu)-S(t-Bu)-W(Boc)-T(Ψ^(Me,Me)pro)-F-C(Trt)-W(Boc)-D(O-t-Bu)-P-GGGK(ivDde)-TGRwas prepared on a 0.2 mmol scale. The ivDde protecting group was removedfrom a 400 mg (nominally 0.08 mmol) portion of the resin (procedure D).The resin was washed with DMF (2×20 mL) and DCM (20 mL), re-suspended inDMF (10 mL) and treated with Fmoc-Adoa (154 mg, 0.4 mmol), HOBt (54 mg,0.4 mmol), DIC (51 mg, 62 μL, 0.4 mmol) and DIEA (139 μL, 0.8 mmol) for4 h. The reagents were filtered off and the resin was washed with DMF(2×20 mL) and DCM (20 mL). The Fmoc group was removed by treatment with20% piperidine in DMF (2×20 mL) (modified procedure C) and the resin waswashed with DMF (2×20 mL) and DCM (20 mL). Coupling with Fmoc-Adoa andFmoc removal were repeated. The resin was re-suspended in DMF (7 mL) andtreated with 3,6,9-trioxaundecane-1,11-dioic acid anhydride solution[prepared by the reaction of the corresponding acid (1.0 g, 0.45 mmol))and DIC (0.56 g, 0.45 mmol) in methylene chloride (5.0 mL) over a periodof 12 hr, the solution was filtered and used directly] for 16 hr. Thereagents were filtered off and the resin was washed with DCM (2×20 mL)and DMF (2×20 mL), re-suspended in DCM (10 mL) and treated with asolution of dipalmitoyl phosphatidyl ethanolamine (690 mg, 1.0 mmol),HATU (450 mg, 1.0 mmol) DIEA (400 mg) in DCM (5.0 mL) and the mixturewas allowed to shake for 26 hr (modified procedure B). The reagents werefiltered off and the resin was washed with DMF (2×20 mL) and DCM (2×20mL) and dried. The resin was then treated with Reagent B (30 mL) for 4hr (procedure G). The resin was filtered off and the filtrate wasconcentrated and treated with 200 mL of anhydrous Et₂O and the crudeproduct was collected as a solid by filtration. This provided 400 mg ofcrude product which was dissolved in DMSO (4.0 mL) after which the pH ofthe solution was adjusted to 7.5 with an aqueous solution ofN-methyl-D-glucamine and stirred 48 h in air to effect formation of thecyclic peptide disulfide. The solution was diluted with water to avolume 40 mL and purified by reverse phase preparative HPLC (Kromasil®Prep C₄, 10μ, 300 Å, 20×250 mm, flow rate 10 mL/min) using a gradient of50-100% water (0.1% TFA)/acetonitrile:MeOH (1:1, 0.1% TFA) over a periodof 15 min. The pure product-containing fractions were collected,combined and lyophilized to afford the target compound (28 mg, 10%yield) as a fluffy white solid.

Examples 17-20 below describe processes for preparing targetedmicrobubbles with fibrin-binding polypeptides conjugated tophospholipids (lipopeptides). Such microbubbles are especially adaptedfor ultrasound imaging.

Example 17 Preparation of Targeted Microbubbles with DSPC/DPPG EnvelopeExample 17A With Comparative Lipopeptide Seq000-PL1

383 mg of a mixture DSPC/DPPG/Seq000-PL1 (molar ratio 47.5/47.5/5,corresponding to 157.5, 148.5 and 77.3 mg of the three components,respectively) and 22.6 g of PEG-4000 were solubilized in 120 g oft-butyl alcohol at 60° C., in a water bath. The solution was filled invials with 0.8 mL of solution each. The samples were frozen at −45° C.and lyophilized. The air in the headspace was replaced with a mixture ofC₄F₁₀/Nitrogen (50/50) and vials capped and crimped. The lyophilizedsamples were reconstituted with 5 mL of H₂O per vial.

Example 17B With Seq005-PL1

Example 17A was repeated, but replacing Seq000-PL1 with the samerelative molar amount of Seq005-PL1.

Example 18 Preparation of Targeted Microbubbles with DPPE/DPPG EnvelopeExample 18A With Comparative Lipopeptide Seq000-PL1

An aqueous suspension of DSPE-PEG1000 (0.5 mg-0.28 μmole) and Seq000-PL1(3.3 mg-0.63 μmole) was prepared in 500 μL of distilled water at 60° C.to obtain a micellar suspension.

Separately, DPPE (15.8 mg-22.8 μmoles) and DPPG (4.2 mg-5.7 μmoles) weredispersed in a solution of PEG4000 10% in distilled water (20 mL) at 70°C. for 20 minutes. The dispersion was then cooled to room temperature.Perfluoroheptane (1.6 mL) was added to the aqueous phase using a highspeed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minuteat 10000 rpm, to obtain an emulsion.

The micellar suspension was mixed with the emulsion and the resultingmixture was heated at 80° C. for 1 hour under agitation. After coolingat room temperature (1 hour), the mixture was washed once bycentrifugation (200 g/10 min—Sigma centrifuge 3K10) to eliminate theexcess of phospholipids. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended withthe initial volume of a 10% PEG 4000 aqueous solution.

The obtained suspension was sampled into DINER vials (1 mL/vial). Thenvials were cooled at −50° C. (Christ Epsilon 2-12DS Freeze Dryer) andfreeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final dryingstep at 30° C. and 0.1 mbar for 7 hours.

The lyophilized product was then exposed to an atmosphere containingC₄F₁₀/Nitrogen (50/50 by volume) and the vials were sealed.

The lyophilized product was dispersed in a volume of water twice theinitial one by gentle hand shaking.

Example 18B With Seq017-PL1

Example 18A was repeated by replacing Seq000-PL1 with the same relativemolar amount of Seq017-PL1.

Example 18C With Seq005-PL1

Example 18A was repeated by replacing Seq000-PL1 with the same relativemolar amount of Seq005-PL1.

Example 19 Preparation of Targeted Microbubbles with DSPC/DSPG EnvelopeExample 19A DSPC/DSPG Formulation with Comparative LipopeptideSeq000-PL1

An aqueous suspension of DSPE-PEG1000 (0.5 mg-0.28 μmole) and Seq000-PL1(3.3 mg-0.63 μmole) was prepared in 500 μL of distilled water at 60° C.to obtain a micellar suspension.

Separately, DSPC (18 mg-22.75 μmoles) and DSPG (2 mg-2.53 μmoles) weredissolved in cyclooctane (1.6 mL) at 80° C. This organic phase was addedto a PEG4000 10% solution in water (20 mL) using a high speedhomogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute at9000 rpm, to obtain an emulsion

The micellar suspension was mixed with the emulsion and the resultingmixture was heated at 80° C. for 1 hour under agitation. After coolingto room temperature (1 hour), the obtained emulsion was washed once bycentrifugation (1500 g/10 min-Sigma centrifuge 3K10) to eliminate theexcess of phospholipids. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended inthe initial volume of a 10% PEG 4000 aqueous solution.

The obtained suspension was sampled into DINER vials (1 mL/vial). Thenvials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) andfreeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final dryingstep at 30° C. and 0.1 mbar for 7 hours.

The lyophilized product was exposed to an atmosphere containingC₄F₁₀/Nitrogen (35/65 by volume) and the vials were sealed.

The lyophilized product was then dispersed in a volume of water twicethan the initial one by gentle hand shaking

Example 19B DSPC/DSPG Formulation with Comparative Peptide Seq000-PL1

Example 19A was repeated, but using 2.6 mg of DSPE-PEG1000 (1.44 μmoles)and 1.9 mg of Seq000-PL1 (0.36 gmole) to prepare the micellarsuspension.

Example 19C DSPC/DSPG Formulation with Seq024-PL1

Example 19A was repeated, but using 2.6 mg Seq024-PL1 (0.45 μmoles) and0.8 mg DSPE-PEG1000 (0.45 μmoles) to prepare the micellar suspension.

Example 19D DSPC/DSPG Formulation with Seq023-PL1

Example 19A was repeated, but using 2.4 mg Seq023-PL1 (0.45 μmoles) and0.8 mg DSPE-PEG1000 (0.45 μmoles) to prepare the micellar suspension.

Example 19E DSPC/DSPG Formulation with Seq016-PL1

Example 19A was repeated, but using 2.3 mg Seq016-PL1 (0.45 μmoles) and0.8 mg DSPE-PEG1000 (0.45 μmoles) to prepare the micellar suspension.

Example 20A-20D Preparation of Targeted Microbubbles with DSPC/StearateEnvelope Example 20A With Comparative Lipopeptide Seq000-PL1

An aqueous suspension of DSPE-PEG1000 (0.5 mg-0.28 μmoles) andSeq000-PL1 12 (3.3 mg-0.63 μmoles) was prepared in 500 μL of distilledwater at 60° C. to obtain the micellar suspension.

Separately, DSPC (18.2 mg-23.1 μmoles) and stearate (1.8 mg-5.8 μmoles)were dispersed in a solution of PEG4000 10% in distilled water (20 mL)at 70° C. for 20 minutes. The dispersion was then cooled to roomtemperature. Perfluoroheptane (1.6 mL) was added to the aqueous phaseusing a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm)for 1 minute at 11000 rpm, to obtain an emulsion.

The micellar solution was mixed with the emulsion and the resultingmixture was heated at 60° C. for 4 hours under agitation. After coolingto room temperature (1 hour), the obtained emulsion was washed once bycentrifugation (200 g/10 min-Sigma centrifuge 3K10) to eliminate theexcess of phospholipids. The separated supernatant (containingemulsified microdroplets of solvent) was recovered and re-suspended withthe initial volume of a 10% PEG 4000 aqueous solution.

The obtained suspension was sampled into DINER vials (1 ml/vial). Thenvials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) andfreeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final dryingstep at 30° C. and 0.1 mbar for 7 hours.

The lyophilized product was then exposed to an atmosphere containingC₄F₁₀/Nitrogen (35/65 by volume) and the vials were sealed.

The lyophilized product was dispersed in a volume of water twice thanthe initial one by gentle hand shaking

Example 20B With Seq017-PL1

Example 20A was repeated by replacing Seq000-PL1 with the same relativemolar amount of Seq017-PL1.

Example 20C With Seq005-PL1

Example 20 A was repeated by replacing Seq000-PL1 with the same relativemolar amount of Seq005-PL1.

Example 20D With Seq016-PL1

Example 20 A was repeated by replacing DSPG with 5.8 μmoles of stearate.

Example 20E-20H Preparation of Targeted Microbubbles with DSPC/DSPAEnvelope Example 20E With Seq005-PL4

DSPC (16.3 mg-20.58 μmoles), DSPA (3.7 mg-5.15 μmoles) andSeq005-PL4(0.26 μmoles, prepared as described above) were dissolved incyclooctane (1.6 mL) at 80° C.

The organic suspension was emulsified in a PEG4000 10% aqueous phase (20mL) using a high speed homogenizer (Polytron PT3000, probe diameter of 3cm) for 1 minute at 8000 rpm to obtain the emulsion.

The resulting emulsion was heated at 80° C. for 1 hour under stirring.After cooling at room temperature (1 hour), the emulsion was washed onceby centrifugation (1500 g/10 min-Sigma centrifuge 3K10) to eliminate theexcess of the phospholipid and the separated supernatants(microdroplets) were recovered and re-suspended in twice the initialvolume of a 10% PEG 4000 aqueous solution.

The emulsion was sampled in DINER vials (1 mL/vial) and then lyophilized(laboratory freeze-dryer Lyobeta-35 TELSTAR) according the followingsequence.

Freezing: 2 h at −50° C.

Main Drying: 12 h at −25° C. and 0.2 mBar

Final Drying: 6 h at 30° C. and 0.1 mBar

Before redispersion, the lyophilisate was exposed to an atmospherecontaining C₄F₁₀/air (50/50 by volume). The lyophilized product was thendispersed in a volume of water twice the initial one by gentle handshaking

Example 20F With Seq005-PL4

DSPC (16.3 mg-20.58 μmoles), DSPA (3.7 mg-5.15 μmoles) andSeq005-PL4(0.795 μmoles, prepared as described above) were dissolved incyclooctane (1.6 mL) at 80° C.

The organic suspension was emulsified in a PEG4000 10% aqueous phase (20mL) using a high speed homogenizer (Polytron PT3000, probe diameter of 3cm) for 1 minute at 8000 rpm to obtain the emulsion.

The resulting emulsion was heated at 80° C. for 1 hour under stirring.After cooling at room temperature (1 hour), the emulsion was washed onceby centrifugation (1500 g/10 min-Sigma centrifuge 3K10) to eliminate theexcess of the phospholipid and the separated supernatants(microdroplets) were recovered and re-suspended in twice the initialvolume of a 10% PEG 4000 aqueous solution.

The emulsion was sampled in DINER vials (1 mL/vial) and then lyophilized(laboratory freeze-dryer Lyobeta-35 TELSTAR) according the followingsequence.

Freezing: 2 h at −50° C.

Main Drying: 12 h at −25° C. and 0.2 mBar

Final Drying: 6 h at 30° C. and 0.1 mBar

Before redispersion, the lyophilisate was exposed to an atmospherecontaining C₄F₁₀/air (50/50 by volume). The lyophilized product was thendispersed in a volume of water twice the initial one by gentle handshaking

Example 20G With Seq005-PL5

DSPC (16.3 mg-20.58 μmoles), DSPA (3.7 mg-5.15 μmoles) andSeq005-PL5(0.26 μmoles, prepared as described above) were dissolved incyclooctane (1.6 mL) at 80° C.

The organic suspension was emulsified in a PEG4000 10% aqueous phase (20mL) using a high speed homogenizer (Polytron PT3000, probe diameter of 3cm) for 1 minute at 8000 rpm to obtain the emulsion.

The resulting emulsion was heated at 80° C. for 1 hour under stirring.After cooling at room temperature (1 hour), the emulsion was washed onceby centrifugation (1500 g/10 min-Sigma centrifuge 3K10) to eliminate theexcess of the phospholipid and the separated supernatants(microdroplets) were recovered and re-suspended in twice the initialvolume of a 10% PEG 4000 aqueous solution.

The emulsion was sampled in DINER vials (1 mL/vial) and then lyophilized(laboratory freeze-dryer Lyobeta-35 TELSTAR) according the followingsequence.

Freezing: 2 h at −50° C.

Main Drying: 12 h at −25° C. and 0.2 mBar

Final Drying: 6 h at 30° C. and 0.1 mBar

Before redispersion, the lyophilisate was exposed to an atmospherecontaining C₄F₁₀/air (50/50 by volume). The lyophilized product was thendispersed in a volume of water twice the initial one by gentle handshaking

Example 20H With Comparative Peptide Seq000-PL2

DSPC (16.3 mg-20.58 μmoles), DSPA (3.7 mg-5.15 μmoles) and Seq000-PL2(0.26 μmoles, prepared as described above) were dissolved in cyclooctane(1.6 mL) at 80° C.

The organic suspension was emulsified in a PEG4000 10% aqueous phase (20mL) using a high speed homogenizer (Polytron PT3000, probe diameter of 3cm) for 1 minute at 8000 rpm to obtain the emulsion.

The resulting emulsion was heated at 80° C. for 1 hour under stirring.After cooling at room temperature (1 hour), the emulsion was washed onceby centrifugation (1500 g/10 min-Sigma centrifuge 3K10) to eliminate theexcess of the phospholipid and the separated supernatants(microdroplets) were recovered and re-suspended in twice the initialvolume of a 10% PEG 4000 aqueous solution.

The emulsion was sampled in DINER vials (1 mL/vial) and then lyophilized(laboratory freeze-dryer Lyobeta-35 TELSTAR) according the followingsequence.

Freezing: 2 h at −50° C.

Main Drying: 12 h at −25° C. and 0.2 mBar

Final Drying: 6 h at 30° C. and 0.1 mBar

Before redispersion, the lyophilisate was exposed to an atmospherecontaining C₄F₁₀/air (50/50 by volume). The lyophilized product was thendispersed in a volume of water twice the initial one by gentle handshaking

Example 21 Dynamic Binding Test of Targeted Microvesicles WithFibrin-Binding Peptides Preparation of Fibrin-Coated Coverslips

Glass coverslips (40 mm in diameter, Bioptechs Inc., Butler, Pa., USA)were coated with fibrin according the following methodology.

Five mL of a solution of BSA 1% w/v in PBS pH 7.4 were added into a 60mm Petri Dish containing one coverslip, and incubated at 37° C. for 15min. Then the coverslip was washed three times with 5 ml of Tween 80/PBS(0.1%, v:v). Twenty five μL of human thrombin solution at 5 U/mL wereadded per mL of human fibrinogen solution (0.5 mg/mL in 50 mM sodiumphosphate, NaCl 280 mM, pH 7.4) and delicately mixed. Five mL of thissolution were immediately distributed in each Petri dish. The coverslipswere incubated for one hour at 37° C. and then dried overnight at 45° C.

Binding Assay

Binding studies of targeted microvesicles were carried out using aparallel-plate flow chamber (FCS2, Bioptech Inc., Butler, Pa., USA) witha chamber gasket of 0.25 mm in thickness, with a customized adapter forupside-down chamber inversion. The coated coverslip was inserted as aplate of the flow chamber. Gas-filled Microvesicles (5×10⁶ bubbles/mL in50% human plasma in PBS) were drawn through the flow chamber using anadjustable infusion pump (Auto Syringe® AS50 Infusion Pump, Baxter,Deerfield, Ill., USA) with a 60 mL syringe (Terumo). The pump flow ratewas adjusted to 1 mL/min to obtain the desired shear rate of about 114s⁻¹. After 10 minutes, the flow was stopped and pictures were takenrandomly on different positions on the coverslip (on surfaces of about0.025 mm²) using a 40× objective and a CCD monochrome camera (F-View II,Soft Imaging Systems, Germany) connected to an inverted Olympus IX 50microscope. The number of microvesicles on each picture was determined,averaged with respect to the total number of pictures and the obtainedvalue was then divided by ten (to obtain the “slope”, i.e. the averageamount of bound microvesicles per minute).

For each preparation of Examples 17-20, the binding assay was repeatedfour times thus obtaining an average value of the slope. The sloperepresents the microvesicle binding rate on the target substrate. Forinstance, a slope value of 8 indicates that an average of eighty (80)microvesicles was bound on the coated coverslip in ten minutes. A higherslope indicates a better capacity of microvesicles to bind to the targetunder flow conditions.

In the following Tables 5-10, the binding activity of the microvesiclesprepared according to Examples 17-20 above is illustrated. As shown inthese Tables, peptides according to the present invention (particularlytheir respective lipopeptides) show superior activities with respect tothe comparative peptide Seq000 (in particular, to correspondinglipopeptides Seq000-PL1 and Seq000-PL2).

TABLE 6 Fibrin- Microvesicle Binding Preparation Lipopeptide of ExampleSlope Seq000-PL1 17A 6.44 Seq005-PL1 17B 8.54

TABLE 7 Fibrin- Microvesicle Binding Preparation Lipopeptide of ExampleSlope Seq000-PL1 18A 4.8 Seq017-PL1 18B 8.9 Seq005-PL1 18C 7.2

TABLE 8 Fibrin- Microvesicle Binding Preparation Lipopeptide of ExampleSlope Seq000-PL1 19A 4.4 Seq000-PL1 19B 4.9 Seq024-PL1 19C 6.2Seq023-PL1 19D 8.5 Seq016-PL1 19E 11.5

TABLE 9 Fibrin- Microvesicle Binding Preparation Lipopeptide of ExampleSlope Seq000-PL1 20A 1.6 Seq017-PL1 20B 3.1 Seq005-PL1 20C 6.1Seq016-PL1 20D 8.4

TABLE 10 Fibrin- Microvesicle Binding preparation Lipopeptide of Exampleslope Seq005-PL4 20E 6.4 Seq005-PL4 20F 8.0 Seq005-PL5 290G 6.0Seq000-PL2 20H 4.6

Example 22 Preparation of the Chelated Complex 1

The synthetic procedure for the preparation of the Chelated Complex 1 isset forth in Scheme 5.

Chelated Complex 1

Fmoc-PAL-PEG-PS resin supported intermediate A prepared as schematizedin FIG. 1 (5.00 g; 0.90 mmol) was shaken in a SPPS vessel with DMAC (40mL) for 1 h to swell the resin. After the solvent was filtered,Fmoc-GGG-OH (1.48 g; 3.60 mmol), HOBt (0.55 g; 3.60 mmol), DIC (0.56 mL;3.60 mmol) and DMAC (40 mL) were added to the resin, the suspensionshaken for 6 h at room temperature, the mixture filtered and the resinwashed with DMAC (5×40 mL). The resin was then shaken with 50%morpholine in DMAC (7 mL) for 10 min, the mixture filtered and fresh 50%morpholine in DMAC (7 mL) was added. The suspension was stirred for 20min then the mixture was filtered and the resin washed with DMAC (5×40mL). Fmoc-Lys(Fmoc)-OH (2.13 g; 3.60 mmol), HOBt (0.55 g; 3.60 mmol),DIC (0.56 mL; 3.60 mmol) and DMAC (40 mL) were added to the resin, thesuspension shaken for 6 h at room temperature, filtered and the resinwashed with DMAC (5×40 mL). The resin was then shaken with 50%morpholine in DMAC (7 mL) for 10 min, the mixture filtered and fresh 50%morpholine in DMAC (7 mL) was added. The suspension was stirred for 20min then the mixture was filtered and the resin washed with DMAC (5×40mL). Fmoc-Lys(Fmoc)-OH (4.26 g; 7.20 mmol), HOBt (1.10 g; 7.20 mmol),DIC (1.12 mL; 7.20 mmol) and DMAC (40 mL) were added to the resin, thesuspension shaken for 6 h at room temperature, filtered and the resinwashed with DMAC (5×40 mL). The resin was then shaken with 50%morpholine in DMAC (7 mL) for 10 min, the mixture filtered and fresh 50%morpholine in DMAC (7 mL) was added. The suspension was stirred for 20min then the mixture was filtered and the resin washed with DMAC (5×40mL). DTPA-Glu (10.7 g; 14.4 mmol), HOBt (2.20 g; 14.4 mmol), DIC (2.26mL; 14.4 mmol), DIEA (4.90 mL; 28.8 mmol) and DMAC (40 mL) were added tothe resin. The suspension was shaken for 24 h at room temperature,filtered and the resin washed with DMAC (5×40 mL), CH₂Cl₂ (5×40 mL) andthen vacuum dried. The resin was shaken in a flask with “Reagent B” (150mL) for 4.5 h. The resin was filtered and the solution was evaporatedunder reduced pressure to afford an oily crude that, after treatmentwith Et₂O (40 mL), gave a precipitate. The precipitate was centrifuged,decanted and washed with Et₂O (4×40 mL) to give a white solid (2.10 g).This product (2.10 g) was dissolved in a mixture of DMSO (36 mL) and H₂O(4.0 mL) and the pH adjusted to 8 with D-(−)-N-methyl glucamine (1.23g). The solution was stirred for 96 hours at room temperature and thenpurified by preparative HPLC. The fractions containing the product werelyophilized to afford the desired chelating ligand (0.280 g; 0.058 mmol)as a white solid. The ligand (0.240 g; 0.050 mmol) was suspended in H₂O(80 mL) and dissolved by addiction of 0.1 N NaOH (8.50 mL; 0.85 mmol) upto pH 6.5. 5.187 mM aq. GdCl₃ (38.3 mL; 0.202 mmol) was addedmaintaining pH 6.5 by means of 0.1 N NaOH (6.0 mL; 0.60 mmol). Thesolution was adjusted to pH 7.0 with 0.1 N NaOH and then loaded onto aXAD 1600 column and eluted with a gradient H₂O/ACN (the desired productelutes with a percentage of ACN=30) to give, after evaporation, compoundthe Chelated complex 1, as sodium salt, (0.167 g; 0.029 mmol) as a whitesolid. Overall yield 3.8%.

Analytical Data for Chelated Complex 1

Mr (Molecular Weight): 5663.73 (C₂₀₅H₂₇₆Gd₄N₄₈Na₁₀O₈₃S₂)

CE (Capillary Electrophoresis): 88.5% (Area %)

MS (Mass Spectroscopy): Obtained data consistent with Chelated Complex 1structure

Example 23 Preparation of the Chelated Complex 2

The synthetic procedure for the preparation of the Chelated Complex 2 isset forth in Scheme 6.

Chelated Complex 2

To Fmoc-PAL-PEG-PS resin supported intermediate B (3.00 g; 0.60 mmol)obtained according to Procedure A, described above, was shaken in a SPPSvessel with DMAC (25 mL) for 1 h to swell the resin. After the solventwas filtered, the resin was washed with DMF (5×25 mL). The resin wasthen shaken with 10% hydrazine in DMF (25 mL) for 15 min, the solventfiltered and fresh 10% hydrazine in DMF (25 mL) was added.

The suspension was stirred for more 20 min then the mixture was filteredand the resin washed with DMF (5×25 mL) and then DMAC (5×25 mL).Fmoc-Lys(Fmoc)-OH (1.42 g; 2.40 mmol), HOBt (0.36 g; 2.40 mmol), DIC(0.37 mL; 2.40 mmol) and DMAC (25 mL) were added to the resin, thesuspension shaken for 24 h at room temperature, filtered and the resinwashed with DMAC (5×25 mL). The resin was then shaken with 50%morpholine in DMAC (25 mL) for 10 min, the mixture filtered and fresh50% morpholine in DMAC (25 mL) was added. The suspension was stirred for20 min then the mixture was filtered and the resin washed with DMAC(5×25 mL). Fmoc-Lys(Fmoc)-OH (2.84 g; 4.80 mmol), HOBt (0.73 g; 4.80mmol), DIC (0.75 mL; 4.80 mmol) and DMAC (25 mL) were added to theresin, the suspension shaken for 24 h at room temperature, filtered andthe resin washed with DMAC (5×25 mL). The resin was then shaken with 50%morpholine in DMAC (25 mL) for 10 min, the mixture filtered and fresh50% morpholine in DMAC (25 mL) was added. The suspension was stirred for20 min then the mixture was filtered and the resin washed with DMAC(5×25 mL). DTPA-Glu (7.17 g; 9.60 mmol), HOBt (1.47 g; 9.60 mmol), DIC(1.50 mL; 9.60 mmol), DIEA (3.26 mL; 9.60 mmol) and DMAC (25 mL) wereadded to the resin. The suspension was shaken for 24 h at roomtemperature, filtered and the resin washed with DMAC (5×25 mL), CH₂Cl₂(5×25 mL) and then vacuum dried. The resin was shaken in a flask with“Reagent B” (100 mL) for 4.5 h. The resin was filtered and the solutionwas evaporated under reduced pressure to afford an oily crude that,after treatment with Et₂O (40 mL), gave a precipitate. The precipitatewas centrifuged, decanted and washed with Et₂O (4×40 mL) to give a whitesolid (1.60 g). This product (1.60 g) was dissolved in a mixture of DMSO(27 mL) and H₂O (3.0 mL) and the pH adjusted to 8 with D-(−)-N-methylglucamine (0.94 g). The solution was stirred for 96 hours at roomtemperature and then purified by preparative HPLC. The fractionscontaining the product were lyophilized to afford the desired chelatingligand (0.260 g; 0.060 mmol) as a white solid. The ligand (0.220 g;0.050 mmol) was suspended in H₂O (80 mL) and dissolved by addiction of0.1 N NaOH (7.40 mL; 0.74 mmol) up to pH 6.5. 6.21 mM aq. GdCl₃ (32.60mL; 0.202 mmol) was added maintaining pH 6.5 by means of 0.1 N NaOH(6.10 mL; 0.61 mmol). The solution was adjusted to pH 7.0 with 0.1 NNaOH and then loaded onto a XAD 1600 column and eluted with a gradientH₂O/ACN (the desired product elutes with a percentage of ACN=30) togive, after evaporation, the Chelated complex 2, sodium salt, (0.174 g;0.033 mmol) as a white solid. Yield 6.6%.

Analytical Data

Mr: 5202.26 (C₁₈₇H₂₄₅Gd₄N₄₃Na₁₀O₇₄S₂)

CE: 87.8% (Area %)

MS: Obtained data consistent with Chelated Complex 2 structure

Example 24 Preparation of the Chelated Complex 4

By following the synthetic procedure of Scheme 6, and by using thesuitable AAZTA ligand instead of the corresponding DTPA ligand theChelated Complex 4 has been also prepared.

Reference Complexes

By starting from the previously known peptide Ac-WQPC*PWESWTFC*WDGGGK-NH₂ provided by WO02/055544 and by following the synthetic procedures ofSchemes 5 and 6, suitably changing the chelating ligand moiety to beconjugated to linker-functionalized peptide intermediate, some chelatedcompounds have been prepared as Reference Compounds for in vivo and invitro tests. The following reference compounds have, in particular, beenprepared:

Example 25 Fibrin MRI Imaging in Human/Mouse Clots

In Vitro MRI Tests

Small plasma clots from different species (human, guinea pig and mouse)ranging from 0.5-7 mm in diameter were formed by combining citrateplasma (1:3,v/v) phospholipids (Reagent Pathromtin) and CaCl2 0.008 M(Dade Behring, Germany) into small 2 ml vials at 37° C. Clots werewashed 3 times with TBS 1× and incubated with contrast agents at 100 μMfor 30 minutes at 37° C. Two clots were prepared for each solution ofincubation. After the incubation clots were washed 4 times with TBS 1×and placed into small vials filled with TBS 1× for MRI analysis.T1-weighted 2D Spin-Echo (SE) images were acquired with the followingparameters: TR/TE=400/8 ms, spatial resolution=344 μm and slicethickness=2 mm. Maximum signal intensity was measured in each imagecontaining clot and compared to the signal from a clots incubated inTBS. After MRI, clots were prepared for ICP analysis.

The Chelated Complex 1 of the invention, Reference compound 1, Referencecompound 2, Reference compound 4, and Reference compound 5 were testedon human clots by MRI at different concentrations in the incubationmedium (25, 100 and 400 μM of complex).

Results

By comparing the signal enhancement in clots registered after incubationwith the compounds at 100 μM concentration, tested compounds could beclassified in term of performance as follows:

TABLE 10 Max signal Enhancement at Comparison to Compound 100 μM ChelateComplex 1 Chelated Complex 1 100%  Reference Compound 2 68% −32%Reference Compound 1 44% −66% Reference Compound 5 41% −69% ReferenceCompound 4 23% −77%

The results indicate that the compound of the invention, ChelatedComplex 1, has the maximum signal enhancement.

Example 26 Fibrin MRI Imaging in Tumor Models

In Vivo MRI Tests:

Three groups of 5 mice each, inoculated with 2.10⁶ neuroblastoma cellswith PBS,

were formed and tested as follows:

Group a: Reference compound 2,

Group b: Chelate Complex 1

Group c: ProHance® (see Protocol)

At day-10 post tumor cells inoculation, at least 4 mice per group withsimilar tumor size (5-10 mm in diameter) were selected for the in vivoMRI tests. After pre-contrast MRI acquisition (T1 and T2 high resolution2D spin-echo and 3D gradient echo images), the test compounds wereadministered (25 μmol/kg iv.) and MRI was performed at 4 h and 24 h postinjection. Maximum signal intensity was measured in the entire tumorarea and in each post contrast image covering the whole tumor and thesignal was compared to the pre-contrast images. At the end of MRI exams,all animals were sacrificed and the tumor, blood, liver, kidneys andfemoral bone were removed and prepared for Gd assay by ICP-AES.

(Inductively coupled plasma atomic emission spectroscopy).

Results

The size of the tumors in all groups was similar (volume of about 500mm³).

A significant tumor signal enhancement was observed 4 h post ChelatedComplex 1 injection whereas with the Reference Compound 2 theenhancement was low and did not cover the whole tumor (<3 slices over 9,˜30% of slices covering the tumor). FIG. 8 shows obtained T1 weightedMRI images, registered at 2T, after injection of 25 μmol/kg of thecomplex. For the ProHance® injection signal distribution was only on theborder and no signal enhancement was observed inside the tumors.

In vitro and in vivo tests described in Examples 25 and 26 wereperformed by following the protocols below.

Protocols

Screening of Fibrin Targeted Compounds of the Invention.

With the aim to test the specificity and efficiency of the compounds ofthe invention the following two protocols have been developed and used.One is dedicated to the in vitro MRI tests on clots and the other forthe in vivo MRI tests on neuroblastoma mouse model.

In Vitro Tests

This protocol has been developed and used to test the specificity andefficiency of the fibrin targeted contrast agents of the invention forsensitive detection of thrombus by use of MRI. The study was performedon clots generated in vitro from different plasma species. Signalenhancement from each sample incubated with the fibrin targeted contrastagent was evaluated and compared to a standard contrast agent ProHance®.

Materials

Test Article

Compounds: The contrast agents tested were compounds of the presentinvention comprising a fibrin peptide, an optional linker and at leastone chelated complex of gadolinium.

Reagents

Compounds: Biological samples (species, biological fluid and strain):

guinea pig plasma(Dunkin Hartley)

mouse plasma CD®-1(ICR)BR IGS

-   -   rabbit plasma (New Zealand White)

Storage: at −20° C.

Test System

The test system used was clots generated in-vitro from different plasmaspecies (Human, guinea pig, mouse and rabbit), chosen because they are asuitable model and easy to replicate for this study.

Equipment

All MRI experiments were performed on a 2T Oxford magnet (bore=45 cmi.d.), equipped with a self-shielded gradient set (16 cm i.d.) driven bysix TECHRON® amplifiers (4:1:1 configuration) with a maximum gradientstrength of 110 mT/m and slew rate of 75 μs, and interfaced to an MRRSconsole (MR Research Systems Ltd, Surrey UK). A bird-cage resonatorantenna (7.3 cm i.d.) was used.

Methods

In-Vitro Tests

The delivered Control Plasma N was obtained from pooled plasma collectedfrom healthy blood donors, stabilized with HEPES buffer solution (12g/L) and lyophilized. Before use, Plasma Control N was reconstituted indistilled water by shaking carefully the suspension to dissolve thelyophilized plasma. Clots were formed, in a 2 mL vial, to which wasadded 300 μl of reconstituted plasma and 300 μL of Pathromtin** SL(silicon dioxide particles, vegetable phospholipids, sodium chloride 2.4g/L, Hepes 14.3 g/L, pH 7.6). The mixture was incubated for 2 minutes at37° C., and then 300 μL of calcium chloride solution, previouslyincubated at 37° C., was added to obtain a new mixture that was furtherincubated for 30 minutes at 37° C. The obtained clot was transferred toa 5 mL tube, washed 3 times with 3 mL of TBS and incubated with the testarticle at the final concentration ranging from 0.0 to 0.2 mM for 30minutes at 37° C. At the end of incubation, clots were rinsed 4 timeswith 3 mL of TBS to remove the unbound test article and then placed in1.5 ml vials for MRI procedure.

High resolution MRI was then performed by using T1-weighted Spin-Echo(SE) sequences. T1 maps were also calculated from the acquisition of 2Dgradient echo images or inversion recovery spin echo images.

At the end of experiment the clots were sent at the AnalyticalLaboratory for ICP-AES analysis.

Assay of Gadolinium in Biological Samples

Apparatus

The assays were carried out on a Jobin-Yvon Mod 24 spectrometeroperating with the following instrumental parameters:

-   -   sample flow: 1 mL/min    -   plasma flame: 6000 to 10000° C.    -   wavelength: 342.247 nm    -   Argon flow: nebulizer 0.3 L/min, transport gas 0.2 L/min,        cooling gas 12 L/min.

Sample digestion was performed by a microwave system (MDS-2000 CEMCorporation).

-   -   Sample preparation and analytical conditions    -   Different preparation for each biological matrix was adopted.    -   Clot solutions were prepared by suspending the clot in 1.5 mL of        nitric acid (65% v/v).    -   1.5 mL of nitric acid (65% v/v) was added to solutions of        incubation (before and after incubation) and to washing        solutions.

The destruction of the organic matrix was performed by subjecting thesamples to a wet ashing process with a microwave oven system.

Finally, the dried residues were dissolved with 3.0 mL of HCL 5% (v/v)and then analysed by ICP-AES.

Data Processing.

Briefly, linearity was evaluated for two standards, low and high,ranging from 0.00 to 20 mg(Gd)/L in HCl 5% (v/v), respectively. Thetotal content of gadolinium in the test sample was calculated by usingthe instrumental calibration straight line and express as μg(Gd)/mL.

MRI Data Analysis

The mean signal intensity was measured within a region of interest (ROI)including a clot, drawn by an operator on each MR image using dedicatedsoftware (MR Research Systems Ltd, Surrey UK). Signal intensityenhancement (Enh %) was determined as:Enh%=100*(SI _(x) −SI ₀)/SI ₀

-   -   where SI₀ and SI_(x) are the mean signal intensity of clots        incubated without and with test articles, respectively.

Protocol for In Vivo Tests

The study aim was to evaluate the specificity and efficiency of fibrintargeted agents of the invention as contrast agents for sensitivedetection of fibrin within solid tumor.

The study was performed on a mouse model of neuroblastoma induced byNeuro-2a cells subcutaneously injected in the right flank of a A/J mouse(see for instance: Y. Chen. Effects of irradiated tumor vaccine andcontinuous localized infusion of granulocyte-macrophagecolony-stimulating factor on neuroblastomas in mice. J. Pediatr Surg.2003. 37(9), 1298-1304; Anthony D. Sandler, Hiroshi Chihara and GenKobayashi. CpG Oligonucleotides Enhance the Tumor Antigen-specificImmune Response of a Granulocyte Macrophage Colony-stimulatingFactor-based Vaccine Strategy in Neuroblastoma Cancer Research 2003, 63,394-399)

Signal enhancement kinetic within tumors was evaluated after injectionof a fibrin targeted contrast agent of the invention and compared to astandard agent, ProHance®.

Materials

Test Article

Compound: The contrast agents tested comprise a fibrin targeted peptide,an optional linker and at least one chelated complex of gadolinium.

Reference Article

Compound: ProHance®

Concentration: 0.5 M

Batch: T2059

Storage: at RT, protected from light

Reagents

Compound: Penicillin/Streptomycin (10000 μg/mL), supplier: Biochrom K G,Berlin, Germany

Compound: L-Glutamine (200 mM), supplier: SIGMA-ALDRICH, Steinheim,Germany

Compound: Foetal bovine serum, supplier: HyClone®, Logan, Utah, USA

Compound: Minimum Essential Medium Eagle, supplier: SIGMA-ALDRICH,Steinheim, Germany

Compound: Dulbecco's Phosphate Buffered Saline (PBS, supplier: SIGMAChemicals, St. Louis, Mo., U.S.A.

Compound: Zoletil 100, supplier: Virbac, Carros, France Compound:Rompun®, supplier: Bayer AG, Laverkusen, Germany

Test System

Animals

Species and strain: mouse A/J (chosen as it is a suitable model forpharmaco-toxicological and imaging studies on neuroblastoma tumor).

-   -   Number and sex of animals: 10 males (5 males/group); 5 animals        for possible replacements    -   Weight and age at arrival: 20-25 g; 5-6 weeks old    -   Supplier: Harlan Italy, S. Pietro al Natisone (UD), Italy

Equipment

All the experiments were performed on a MRRS console (MR ResearchSystems Ltd, Surrey UK) interfaced to a 2T Oxford magnet (bore=45 cmi.d.), equipped with a 16 cm i.d. self-shielded gradient set, driven bysix TECHRON® amplifiers (4:1:1 configuration) with a maximum gradientstrength of 110 mT/m and slew rate of 75 μs. As R. F. coil, a quadratureresonator optimized to the mouse size was used.

Methods

Experimental Design

Mouse neuroblastoma cell line (BS TCL 128 Neuro-2a) was supplied by theIstituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia,Brescia. The cells were grown in 90% MEM medium and 10% fetal bovineserum, collected, washed two times with physiological saline andresuspended in PBS (10⁶ cells/0.2 mL). 10⁶ cells were injectedsubcutaneously in the right flank of each animal.

The tumor development was followed every other day after inoculation bymeasuring the tumor diameter until the day of the MRI experiment.Animals with a tumor diameter ranging from 300 to 700 mm were selectedfor in vivo imaging. Signal enhancement of the tumor was followed by MRIup to 24 h after the administration of the test article and it wascompared with that of the reference article. Animals were anaesthetizedand during the experimental phase the anesthesia was maintained.

The test articles and ProHance® (reference article) were administered atdoses in the range of 0.025-0.1 mmol/kg.

High resolution T1-weighted Spin-Echo and Gradient Echo sequences wereused to achieve images with a suitable contrast to detect tumors and tofollow the contrastographic effects of test and reference articles.

At the end of the experiment, animals were sacrificed. Tumor, blood,kidneys, liver and femoral bones were then taken, weighed and stored atabout 4° C. until analyzed by ICP-AES.

Assay of Gadolinium in Biological Samples

Apparatus

The assays was carried out on a Jobin-Yvon Mod 24 spectrometer operatingwith the following instrumental parameters:

-   -   sample flow: 1 mL/min    -   plasma flame: 6000 to 10000° C.    -   wavelength: 342.247 nm    -   Argon flow: nebulizer 0.3 L/min, transport gas 0.2 L/min,        cooling gas 12 L/min.    -   Sample digestion was performed by a microwave system (MDS-2000        CEM Corporation).

Sample Preparation and Analytical Conditions

Different preparation for each biological matrix was adopted:

-   -   Tumor solutions were prepared by suspending the tumor,        accurately weighed, in 1.5 mL of nitric acid (65% v/v).

Liver was prepared by measuring the liver, accurately weighed, in 1.5 mLof nitric acid (65% v/v).

Kidney solutions were prepared by suspending each kidney, accuratelyweighed, in 1.5 mL of nitric acid (65% v/v).

Blood solutions were prepared by mixing 1 mL of blood in 1.5 mL ofnitric acid (65% v/v).

Bone solutions were prepared by suspending each femur, accuratelyweighed, in 1.5 mL of nitric acid (65% v/v).

The destruction of the organic matrix was performed by subjecting thesamples to a wet ashing process with a microwave oven system.

Finally the dried residues were dissolved with 3.0 mL of HCL 5% (v/v)and then analysed by ICP-AES.

Data Processing.

Briefly, linearity was evaluated for two standards, low and high,ranging from 0.00 to 20 mg(Gd)/L in HCl 5% (v/v), respectively. Thetotal content of gadolinium in the test sample was calculated by usingthe instrumental calibration straight line and express as μg(Gd)/mL.

MRI Data Analysis

The mean signal intensity was measured within a region of interest (ROI)including the entire tumor, drawn by an operator on each MR image usingdedicated software (MR Research Systems Ltd, Surrey UK). Signalintensity enhancement (Enh %) was determined as:Enh%=100*(SI _(x)(t)−SI ₀)/SI ₀

where SI₀ and SI_(x)(t) are the mean signal intensity of tumor pre andpost injection of the contrast agent (test articles or reference),respectively.

Data collected (bodyweight, clinical signs, gross pathology examination)were subjected to qualitative analysis.

Representative Embodiments

The following non-limiting, enumerated embodiments further describe thepresent invention:

-   1. An isolated fibrin-binding peptide having an amino acid sequence    selected from the group consisting of the sequences provided in    Table 1 or Table 2.-   2. A diagnostic imaging agent comprising a fibrin-binding peptide    according to embodiment 1, wherein said fibrin-binding peptide is    linked to a detectable label.-   3. The imaging agent of embodiment 2, wherein said fibrin-binding    peptide is linked to at least one paramagnetic metal atom suitable    for magnetic resonance imaging, optionally via a chelator.-   4. The imaging agent of embodiment 2, wherein said fibrin-binding    peptide is linked to an echogenic label suitable for ultrasound    imaging.-   5. The imaging agent of embodiment 2, wherein the fibrin binding    peptide is linked to a diagnostic radionuclide, optionally via a    chelator-   6. The imaging agent of embodiment 5, wherein the diagnostic    radionuclide is selected from the group consisting of ⁶⁴Cu, ⁶⁷Ga,    ⁶⁸Ga, ^(99m)Tc, and ¹¹¹In.-   7. The imaging agent of embodiment 2, wherein said fibrin-binding    peptide is fluoresceinated.-   8. A therapeutic agent comprising a fibrin-binding peptide according    to embodiment 1, wherein said fibrin-binding peptide is linked to a    therapeutic agent.-   9. The therapeutic agent of embodiment 8, wherein the fibrin-binding    peptide is linked to a therapeutic radionuclide, optionally via a    chelator.-   10. The therapeutic agent of embodiment 9, wherein the therapeutic    radionuclide is selected from the group consisting of ⁶⁴Cu, ⁹⁰Y,    ¹⁰⁵Rh, ¹¹¹In, ¹¹⁷mSn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb,    ¹⁷⁷Lu, ¹⁸⁶⁻¹⁸⁸Rc, and ¹⁹⁹Au.-   11. The imaging agent of embodiment 3, wherein the chelator is    selected from the group consisting of DTPA, DTPA-GLU, DTPA-Lys,    DOTA, AAZTA, and the following derivatives thereof:

-   12. The imaging agent of embodiment 3, selected from the group    consisting of:

-   13. The imaging agent of embodiment 4, wherein the echogenic label    suitable for ultrasound imaging comprises a microballoon comprising    a gas.-   14. The imaging agent of embodiment 4, wherein the echogenic label    suitable for ultrasound imaging comprises a microbubble comprising a    gas.-   15. The imaging agent of embodiment 14, wherein the microbubble    comprises a mixture of two or more selected form the group    consisting of DSPC, DPPG, DPPA, DSPE-PEG1000, DSPE-PEG2000,    DSPE-PEG3400, DPPE-PEG3400, palmitic acid and stearic acid.-   16. An ultrasound contrast agent comprising a microbubble comprising    a gas, wherein the microbubble comprises a lipopeptide comprising a    fibrin-binding peptide of embodiment 1 linked to a phospholipid.-   17. The ultrasound contrast agent of embodiment 16 comprising a    lipopeptide selected from the group consisting of: Seq000-PL1,    Seq005-PL1, Seq016-PL1, Seq017-PL1, Seq023-PL1 and Seq024-PL1.-   18. The ultrasound contrast agent of embodiment 17, wherein the    microbubble further comprises DSPC and DPPG.-   19. The ultrasound contrast agent of embodiment 17, wherein the    microbubble further comprises DSPE-PEG1000, DPPE and DPPG.-   20. The ultrasound contrast agent of embodiment 17, wherein the    microbubble further comprises DSPE-PEG1000, DSPC and DSPG.-   21. The ultrasound contrast agent of embodiment 17, wherein the gas    comprises SF₆ or a perfluorocarbon, optionally in admixture with    air, nitrogen, oxygen or carbon dioxide.-   22. The ultrasound contrast agent of any one of embodiments 18 to    20, wherein the gas comprises C₄F₁₀, optionally in admixture with    air, nitrogen, oxygen or carbon dioxide.-   23. The ultrasound contrast agent of embodiment 16, further    comprising a lyophilization additive and/or a bulking agent.-   24. An isolated fibrin-binding peptide having an amino acid sequence    Ac-WQPC*PWESWTFC*WDPGGGK- NH₂ (SEQ ID NO. 2), in which one or more    of the following modifications have been made:

(a) replacement of Trp⁶ with Ala;

(b) replacement of Phe¹¹ with Cha; and

(c) addition of one or more polar amino acids at the N terminus.

-   25. A diagnostic imaging agent comprising a fibrin-binding peptide    of embodiment 24, wherein said fibrin-binding peptide is linked to a    detectable label.-   26. A therapeutic agent comprising a fibrin-binding peptide of    embodiment 24, wherein said fibrin-binding peptide is linked to a    therapeutic agent.-   27. A compound of general Formula (I)    A[-Y(-T)_(r)]_(s)  (I)

wherein

A is a fibrin-binding peptide moiety comprising an amino acid sequenceselected from the group consisting of the sequences provided in Table 1or Table 2;

Y is a suitable linking moiety connecting A with at least one T; when sis 2, the units Y may be the same or different from each other;

T is, independently in each occurrence, a diagnostically ortherapeutically active moiety;

s is 1 or 2, and

r is, independently in each occurrence, an integer from 1 to 8;

-   -   or a physiologically acceptable salt thereof.

-   28. A compound of embodiment 27, wherein T is a diagnostically    active moiety.

-   29. A compound of embodiment 28, wherein the diagnostically active    moiety is selected from the group consisting of a chelated gamma ray    or positron emitting radionuclide, a paramagnetic metal ion in the    form of a chelated or polychelated complex, an X-ray absorbing agent    including an atom of atomic number higher than 20, a dye molecule, a    fluorescent molecule, a phosphorescent molecule, a molecule    absorbing in the UV spectrum, a quantum dot, a molecule capable of    absorption within near or far infrared radiations, and a moiety    detectable by ultrasound.

-   30. A compound of embodiment 27, wherein T is a therapeutically    active moiety.

-   31. A compound of embodiment 30, wherein the therapeutically active    moiety is selected from the group consisting of a thrombolytic    agent, a fibrinolytic agent, a cytotoxic agent, an agent for    selective killing and/or inhibiting the growth of tumor cells and a    radiotherapeutic agent.

-   32. A compound of embodiment 27, wherein Y is selected from the    group consisting of a linear or branched divalent linking moiety and    a linear or branched polyfunctional linking moiety.

-   33. A compound of embodiment 32, wherein the divalent linking moiety    comprises—GGGK.

-   34. A compound of embodiment 27 wherein A comprises Seq005 as shown    in Table 1.

-   35. An intermediate compound comprising a peptide moiety A    conjugated with an optionally protected Y moiety or with an    optionally protected, sub-unit of a Y moiety, wherein A is a    fibrin-binding peptide having an amino acid sequence selected from    the group consisting of the sequences provided in Table 1 or Table 2    and Y is a linking moiety.

1. An isolated fibrin-binding peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOS:1 to 16, SEQ IDNOS:19-86, SEQ ID NOS:88-94, SEQ ID NOS:96-121 and SEQ ID NOS:134-135.2. An isolated fibrin-binding peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOS:1-2 and SEQ ID NOS:134-135.
 3. A pharmaceutical composition comprising a fibrin-bindingpeptide of claim 1 or a pharmaceutically acceptable salt thereof.
 4. Thepharmaceutical composition of claim 3, further comprising apharmaceutically acceptable ingredient, excipient, carrier, adjuvant orvehicle.
 5. A diagnostic imaging agent comprising at least one of thefibrin-binding peptides recited in claim 1, wherein said fibrin bindingpeptide is linked to a detectable label, optionally via a linker or aspacer.
 6. The diagnostic imaging agent of claim 5, wherein said agentis adapted for use in a modality selected from the group consisting of:magnetic resonance imaging, nuclear imaging, ultrasound imaging, x-rayimaging, and optical imaging.
 7. The diagnostic imaging agent of claim6, wherein said agent is adapted for use in optical imaging.
 8. Thediagnostic imaging agent of claim 7, wherein the detectable label isselected from the group consisting of dye molecules, fluorescentmolecules, phosphorescent molecules, molecules absorbing in the UVspectrum, a quantum dot, and molecules capable of absorption within nearor far infrared radiations.
 9. The diagnostic imaging agent of claim 8,wherein the fibrin binding peptide is linked to the detectable label viaa linker.
 10. The diagnostic agent of claim 8, wherein the detectablelabel is a molecule capable of absorption within near infrared radiationselected from the group consisting of Cy5.5, IRDye800, indocyanine green(ICG), tetrasulfonic acid substituted indocyanine green (TS-ICG), andcombinations thereof.
 11. The diagnostic agent of claim 8, wherein thedetectable label is a dye molecule consisting of an organic chromophoreor fluorophore having extensive delocalized ring systems and havingabsorption or emission maxima in the range of 400-1500 nm.
 12. Thediagnostic imaging agent of claim 8, wherein the detectable label is afluorescent molecule.
 13. The diagnostic imaging agent of claim 5,wherein said agent is adapted for use in ultrasound imaging.
 14. Thediagnostic imaging agent of claim 5, wherein said agent comprisesgas-filled microvesicles.
 15. The diagnostic imaging agent of claim 14,wherein said microvesicles comprise gas-filled microbubbles.
 16. Apeptide-phospholipid conjugate comprising a fibrin-binding peptide ofclaim 1 and a phospholipid.
 17. The peptide-phospholipid conjugate ofclaim 16 wherein the phospholipid is selected from the group consistingof: distearoyl phosphatidyl-ethanolamine (DSPE) and dipalmitoylphosphatidylethanolamine (DPPE).
 18. The peptide-phospholipid conjugateof claim 16 wherein the phospholipid is pegylated.
 19. Thepeptide-phospholipid conjugate of claim 18 wherein the pegylatedphospholipid is selected from the group consisting of: distearoylphosphatidyl-ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) anddipalmitoyl phosphatidylethanolamine-polyethylene glycol 2000(DPPE-PEG2000).
 20. The peptide-phospholipid conjugate of claim 16wherein the peptide and phospholipid are attached by a linking group.21. The peptide-phospholipid conjugate of claim 20 wherein the linkinggroup comprises a moiety selected from the group consisting of:hydrophilic polymers and an amino acid chain.
 22. Thepeptide-phospholipid conjugate of claim 21 wherein the hydrophilicpolymer is polyethylenglycol.
 23. The peptide-phospholipid conjugate ofclaim 21 wherein the amino acid chain comprises proline.
 24. Anultrasound contrast agent comprising a peptide-phospholipid conjugate ofclaim
 16. 25. The ultrasound contrast agent of claim 24, wherein thecontrast agent comprises a gas-filled microvesicle.
 26. The ultrasoundcontrast agent of claim 25, wherein the gas filled microvesiclecomprises a phospholipid.
 27. The ultrasound contrast agent of claim 26,wherein the phospholipid is selected from the group consisting of:distearoyl-phosphatidylcholine (DSPC), dipalmitoyl-phosphatidylglycerol(DPPG), DPPE, distearolyphosphatidylglycerol (DSPG), and distearoylphosphatidic acid (DSPA).
 28. The ultrasound contrast agent of claim 25,wherein the gas-filled microvesicle comprises two or more componentsselected form the group consisting of: DSPC, DPPG, dipalmitoylphosphatidic acid (DPPA), DSPA, DPPE, DSPG, polethyleneglycol1000(DSPE-PEG1000) DSPE-PEG2000, palmitic acid and stearic acid.
 29. Theultrasound contrast agent of claim 26, wherein the gas is selected fromthe group consisting of: air, nitrogen, oxygen, CO₂, hydrogen, nitrousoxide, a noble or inert gas, a radioactive gas, a hyperpolarized noblegas, a fluorinated gas, a low molecular weight hydrocarbon, acycloalkane, an alkene, an alkyne, an ether, a ketone, an ester, ahalogenated gas and mixtures thereof.
 30. The ultrasound contrast agentof claim 29, wherein the gas comprises a fluorinated gas.
 31. Theultrasound contrast agent of claim 30, wherein the gas is a mixture ofC₄F₁₀ and nitrogen.
 32. The ultrasound contrast agent of claim 26further comprising a therapeutic agent.
 33. An ultrasound contrast agentcomprising at least one fibrin-binding peptide of claim
 1. 34. Theultrasound contrast agent of claim 33, wherein the contrast agentcomprises a gas filled microvesicle.
 35. The ultrasound contrast agentof claim 34, wherein the gas filled microvesicle comprises aphospholipid.
 36. The ultrasound contrast agent of claim 35, wherein thephospholipid is selected form the group consisting ofdilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine(“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”),diarachidoylphosphatidylcholine (“DAPC”), DSPC,1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”),1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”),1-palmitoyl-2-stearoylphosphatidylcholine (“PSPC”),1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”),dioleoylphosphatidylycholine (“DOPC”), 1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts,diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts,dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts,DPPG and its alkali metal salts, DSPG and its alkali metal salts,dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts,dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts,dipalmitoyl phosphatidic acid DPPA and its alkali metal salts, DSPA,diarachidoyl phosphatidic acid (“DAPA”) and its alkali metal salts,dimyristoyl phosphatidyl-ethanolamin-e (“DMPE”), DPPE, DSPE, dimyristoylphosphatidylserine (“DMPS”), diarachidoyl phosphatidylserine (“DAPS”),dipalmitoyl phosphatidylserine (“DPPS”), distearoylphosphatidylserine(“DSPS”), dioleoylphosphatidylserine (“DOPS”), dipalmitoyl sphingomyelin(“DPSP”), and distearoyl sphingomyelin (“DSSP”).
 37. The ultrasoundcontrast agent of claim 35, wherein the phospholipid is selected fromthe group consisting of: DSPC, DPPG, DPPE, DSPG, and DSPA.
 38. Theultrasound contrast agent of claim 34, wherein the gas-filledmicrovesicle comprises two or more components selected form the groupconsisting of: DSPC, DPPG, DPPA, DSPA, DPPE, DPPG, DSPE-PEG1000,DSPE-PEG2000, palmitic acid and stearic acid.
 39. The ultrasoundcontrast agent of claim 34, wherein the gas is selected from the groupconsisting of air, nitrogen, oxygen, CO₂, hydrogen, nitrous oxide, anoble or inert gas, a radioactive gas, a hyperpolarized noble gas, afluorinated gas, a low molecular weight hydrocarbon, a cycloalkane, analkene, an alkyne, an ether, a ketone, an ester, a halogenated gas andmixtures thereof.
 40. The ultrasound contrast agent of claim 34, whereinthe gas comprises a fluorinated gas.
 41. The ultrasound contrast agentof claim 40, wherein the gas is a mixture of C₄F₁₀ and nitrogen.
 42. Theultrasound contrast agent of claim 34, further comprising a therapeuticagent.
 43. A composition comprising at least one of the fibrin-bindingpeptides recited in claim 1 and a therapeutic agent.
 44. A diagnosticimaging method or a method of treatment of clots or thromboembolicdisease comprising administering to a mammal a composition comprising atleast one of the fibrin-binding peptides recited in claim
 1. 45. Thediagnostic imaging or treatment method of claim 44, wherein thecomposition further comprises a detectable label or therapeutic agent.46. The diagnostic imaging or treatment method of claim 44, wherein thedetectable label is selected from the group consisting of an opticalimaging agent and an ultrasound contrast agent.
 47. The diagnosticimaging or treatment method of claim 44, wherein the compositioncomprises a therapeutic agent selected from the group consisting of:anticoagulant-thrombolytic or fibrinolytic agents capable of clotslysis, anti-angiogenic agents, cytotoxic agents, and radiotherapeuticagents.
 48. A method for diagnostic imaging or treating pathologicalconditions associated with clots or thromboembolic disease comprisingadministering to a mammal a composition comprising at least one of thefibrin-binding peptides recited in claim
 1. 49. The method of claim 48,wherein the composition further comprises a detectable label ortherapeutic agent.
 50. The diagnostic imaging or treatment method ofclaim 49, wherein the detectable label comprises an agent selected fromthe group consisting of an ultrasound contrast agent and an opticalimaging agent.
 51. The treatment method of claim 49, wherein thecomposition comprises a therapeutic agent selected from the groupconsisting of: anticoagulant-thrombolytic or fibrinolytic agents capableof clots lysis, anti-angiogenic agents, cytotoxic agents,chemotherapeutic agents, tumoricidal agents, and radiotherapeuticagents.
 52. A method for imaging fibrin-containing tissue in a mammalcomprising administering an effective amount of a composition of claim 5to the mammal and imaging the mammal.
 53. A method for imagingfibrin-containing tissue in a mammal comprising administering aneffective amount of a composition of claim 33 to the mammal andsubjecting the mammal to ultrasound scanning to image saidfibrin-containing tissue.
 54. The fibrin-binding peptide of claim 1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 1-16, SEQ ID NOS:19-34, SEQ ID NOS:37-40, SEQ ID NOS:47-50,SEQ ID NOS:55-56, SEQ ID NOS:63-74, SEQ ID NOS:77-86, SEQ ID NOS:88-91,SEQ ID NOS:93-94, SEQ ID NOS:96-104, SEQ ID NOS:106-107, SEQ ID NO:109,SEQ ID NOS:111-115, and SEQ ID NOS:117-121.
 55. A diagnostic imagingagent comprising at least one of the fibrin-binding peptides recited inclaim 54, wherein said fibrin binding peptide is linked to a detectablelabel, optionally via a linker or spacer.
 56. The diagnostic imagingagent of claim 55, wherein the agent is adapted for use in a modalityselected from ultrasound imaging and optical imaging.
 57. Thepeptide-phospholipid conjugate of claim 16, selected from the groupconsisting of SEQ ID NO: 123, SEQ ID NOS:125-128 and SEQ ID NOS:132-133.